CN115598231A - Chromatographic method for quantifying nonionic surfactant in composition containing nonionic surfactant and polypeptide - Google Patents

Abstract

The present invention relates to chromatographic methods for quantifying nonionic surfactant in compositions comprising nonionic surfactant and polypeptide. In particular, the present invention provides methods for quantifying a nonionic surfactant in a composition comprising a polypeptide and a nonionic surfactant, wherein the quantification exhibits reduced interference between the nonionic surfactant and the polypeptide. Also provided are methods wherein the compositions further comprise N-acetyltryptophan and the quantification exhibits reduced interference between the nonionic surfactant, the polypeptide, and the N-acetyltryptophan.

Description

Chromatographic method for quantifying nonionic surfactant in composition containing nonionic surfactant and polypeptide

The divisional application of PCT application PCT/US2017/046725 entitled "chromatography method for quantifying nonionic surfactant in composition comprising nonionic surfactant and polypeptide" filed on 8/14 in 2017, which entered the chinese national phase date of 20/3 in 2019, application No. 201780057815.5.

Cross Reference to Related Applications

This application claims priority to U.S. provisional patent application No. 62/375,373, filed 2016, 8, 15, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

The present invention provides methods for analyzing polypeptide formulations for the presence of polysorbates.

Background

Polysorbate 20 (PS 20) is a surfactant commonly used in polypeptide formulations to protect products from physical damage during processing and storage (Kerwin, b.,2007, j.pharm.sci., 97 (8): 2924-2935). Due to its importance for product stability, PS20 must be accurately quantified in the control system of each product. PS20 can be quantified by spectrophotometry, fluorescence micelle assay or high performance liquid Chromatography-evaporative light scattering detector (HPLC-ELSD) assay (see, e.g., kim, J.and Qiu, J., analytical chip acta 806, 144-151, 2014, hewitt et al, journal of Chromatography A,1215 (1): 156-160, 2008).

The Evaporative Light Scattering Detector (ELSD) assay is preferred as a control system assay because it does not require long conditioning times relative to fluorescent micelle assays. The ELSD process can also avoid the need to use the same polysorbate batch as the standard curve preparation used in the production. In addition, fluorescent micellar assays are susceptible to interference by non-specific proteins, particularly for hydrophobic proteins and Antibody Drug Conjugates (ADCs). The vcMMAE linker-drug of the ADC introduces additional hydrophobicity to the protein, which may lead to increased protein interference when quantifying PS 20. In some cases, this non-specific protein interference can be mitigated by using HPLC-ELSD assays.

Although the HPLC-ELSD assay can reduce the extent of protein interference, this interference is not completely eliminated. The problem of protein interference becomes particularly problematic at oligosorbitol ester concentrations and when the protein is more hydrophobic and/or concentrated. In addition, the effect of protein interference depends largely on the column resin batch used. Strategies to alleviate these problems include a) spiking PS20 to dilute protein interference without reducing PS20 reaction, and b) removing protein from the sample by protein precipitation.

The spiking method requires dilution of the sample with the PS20 stock solution at the formulation target concentration. This sample preparation diluted the protein concentration while keeping the PS20 concentration approximately constant. The amount of PS20 spiked into the sample was then subtracted during data analysis. Because the relationship between the ELSD response and the mass analyzed in the detector follows a power law, spiking in PS20 disproportionately reduces the contribution of the protein to the ELSD signal. In some cases, the spiking method has been shown to improve the accuracy of PS20 quantification, but in cases where this is not a viable solution, protein precipitation must be used. Although it is effective in removing protein interference, the HPLC-ELSD precipitation method is not ideal due to overnight sample preparation time, large sample volume and variability in sample preparation. In contrast, the same HPLC-ELSD conditions were used for protein removal, but without an important sample preparation procedure. What is needed is a more robust solution to eliminate protein interference and produce consistent PS20 quantitation under all chromatographic conditions.

All references, including patent applications and publications, cited herein are incorporated by reference in their entirety.

Disclosure of Invention

In some aspects, the present invention provides a method for quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide, wherein interference between the nonionic surfactant and the polypeptide is reduced during the quantifying, wherein the method comprises the steps of a) applying the composition onto a mixed mode anion exchange chromatography material, wherein the composition is applied onto the chromatography material in a solution comprising a mobile phase a and a mobile phase B, wherein the mobile phase a comprises an aqueous solution of an acid and the mobile phase B comprises a methanol solution of an acid, wherein the polypeptide specifically and non-specifically binds to the chromatography material; b) Eluting the specifically bound polypeptide from the mixed mode anion exchange chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step a); c) Eluting the non-ionic surfactant and non-specifically bound polypeptide from the chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step c); d) Quantifying the nonionic surfactant, wherein interference between the nonionic surfactant and the polypeptide during the quantifying is reduced. In some embodiments, the ratio of mobile phase B to mobile phase a in step a) is about 10. In some embodiments, in step B), the ratio of mobile phase B to mobile phase a is increased to about 40. In some embodiments, in step c), the ratio of mobile phase B to mobile phase a is increased to about 100. In some embodiments, mobile phase a comprises an aqueous solution of about 2% acid. In some embodiments, mobile phase B comprises about 2% acid in methanol. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid.

In some embodiments, the flow rate for chromatography is about 1.25 mL/min. In some embodiments, step b) begins about 1 minute after the start of chromatography and ends about 3.4 minutes after the start of chromatography.

In some embodiments, step c) begins about 3.5 minutes after chromatography begins and ends about 4.6 minutes after chromatography begins. In some embodiments, the nonionic surfactant is a poloxamer (P188) or a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the nonionic surfactant in the composition is from about 0.001% to 1.0% (w/v). In some embodiments, the concentration of protein in the composition is from about 1mg/mL to about 250mg/mL. In some embodiments, the formulation has a pH of about 4.5 to about 7.5. In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is suitable for administration to a subjectThe pharmaceutical preparation of (1). In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody (glycoengineered antibody), antibody fragment, antibody drug conjugate, THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column. In some embodiments, the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is

MAX chromatography materials. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD).

In some aspects, the invention provides a method for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a mobile phase a and a mobile phase B, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises a solution of ammonium hydroxide in an organic solvent; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step a); c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step c); d) The nonionic surfactant was quantified. In some cases In an embodiment, the organic solvent of mobile phase B is methanol. In some embodiments, the ratio of mobile phase B to mobile phase a in step a) is about 10. In some embodiments, in step B), the ratio of mobile phase B to mobile phase a is increased to about 45. In some embodiments, in step c), the ratio of mobile phase B to mobile phase a is increased to about 100. In some embodiments, mobile phase a comprises about 2% aqueous ammonium hydroxide. In some embodiments, mobile phase B comprises about 2% methanolic ammonium hydroxide solution. In some embodiments, the flow rate for chromatography is about 1.4 mL/min. In some embodiments, step b) begins about 1 minute after the start of chromatography and ends about 4.4 minutes after the start of chromatography. In some embodiments, step c) begins about 4.5 minutes after chromatography begins and ends about 7.6 minutes after chromatography begins. In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is about 0.001% to 1.0% (w/v). In some embodiments, the organic solvent of mobile phase B is acetonitrile. In some embodiments, the ratio of mobile phase B to mobile phase a in step a) is about 10. In some embodiments, step B) increases the ratio of mobile phase B to mobile phase a to about 40. In some embodiments, the ratio of mobile phase B to mobile phase a in step c) is increased to 100. In some embodiments, mobile phase a comprises about 2% aqueous ammonium hydroxide or 43% methanol solution. In some embodiments, mobile phase B comprises about 2% ammonium hydroxide in acetonitrile. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of poloxamer in the composition is between about 0.001% and 1.0% (w/v). In some embodiments, the composition further comprises N-acetyltryptophan and/or methionine. In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM. In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM. In some embodiments The concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL. In some embodiments, the formulation has a pH of about 4.5 to about 7.5. In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate Thiomab ^TM ，THIOMAB ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD).

Drawings

Figure 1 is a superposition of ADC without PS20 and 0.6mg/ml PS20 standard using multistep gradients with 5% methanol addition. The elution solvent contained 2% formic acid.

Figure 2 shows a comparison of methanol multi-step gradient experiments and isopropanol step gradient experiments (both containing 2% formic acid) on different cartridges. For each stack, there is an A1 ADC without PS20 on the first column (trace 1), an A1 ADC without PS20 on the second column (trace 2) and a PS20 standard (trace 3).

Figure 3 shows the results of the experimental method optimization design. The solid line shows the directionality of the statistical model fitted to the data according to the JMP10 software. The dashed lines defining each solid line represent the error associated with the fit. The slope of the line represents the effect of each factor on the PS20 peak area and PS20 peak width.

Figure 4 shows a comparison of PS20 method chromatograms from DoE method optimization using a 40% meoh wash and a 50% meoh wash. For these experiments, the flow rate was 1.25mL/min, 12. Mu.g of PS20 was loaded, and the wash duration was 3 minutes.

FIG. 5 shows 10mg/ml A10 ADC without PS20 and 0.6mg/ml PS20 standard in mobile phase using 0.2% trifluoroacetic acid.

FIG. 6 shows 20mg/ml A1 ADC without PS20 and 0.6mg/ml PS20 standard in mobile phase using 2% formic acid.

FIG. 7 shows 20mg/ml A1 ADC without PS20 and 0.7mg/ml PS20 standard in mobile phase using 2% acetic acid.

FIG. 8 shows a comparison between mobile phases containing formic acid and acetic acid using water (trace 1), A1 ADC without PS20, 20mg/mL (trace 2) and 0.2mg/mL PS20 doped with A1 ADC,20mg/mL (trace 3).

FIG. 9 shows the use of methanol/acetic acid elution in the difference

Various maps of PS20 standards run on MAX cartridges. A typical map (trace 1), with a trailing peak (trace 2), shows a split peak (trace 3) and a peak with a slight trailing (trace 4). This pattern variability does not affect the quantification of standards, controls or protein samples.

Figure 10 shows the MeOH/acetic acid process. Typical 20 μ L of water for injection (trace 1), A1 ADC formulation buffer without PS20 (trace 2), A1 ADC without PS20 (trace 3) and the lowest PS20 standard of 0.1mg/mL (trace 4).

FIGS. 11A and 11B show evaluation of A16/A17 without PS20 using method 1 of example 1. FIG. 11A shows ELSD and FIG. 11B shows UV (280 nm). Water (trace 1), a16/a17 formulation buffer without PS20 (trace 2) and a16/a17 protein without PS20 (trace 3) were evaluated using method 1 of example 1.

FIGS. 12A and 12B show ELSD chromatograms of different A16/A17 buffer compositions using the method of example 1. FIG. 12A shows buffers with NAT 20mM histidine-HCl, 1mM NAT,5mM methionine, 240mM sucrose (trace 1); 20mM histidine-HCl, 1mM NAT,240mM sucrose (trace 2); 20mM histidine-HCl, 5mM NAT (trace 3). FIG. 12B shows NAT-free buffers 20mM histidine-HCl, 5mM methionine, 240mM sucrose (trace 1); 20mM histidine-HCl, 25mM methionine (trace 2); 20mM histidine-HCl (trace 3). 50 μ L of buffer was injected.

FIGS. 13A and 13B show ELSD chromatograms of different buffer compositions using a modified method (MCX cartridge and ammonium hydroxide in mobile phase). FIG. 13A shows buffers with NAT 20mM histidine-HCl, 1mM NAT,5mM methionine, 240mM sucrose (trace 1); 20mM histidine-HCl, 1mM NAT,240mM sucrose (trace 2); 20mM histidine-HCl, 5mM NAT (trace 3); and PS 20-free protein with NAT (trace 4). FIG. 13B shows NAT-free buffers 20mM histidine-HCl, 5mM methionine, 240mM sucrose (trace 1); 20mM histidine-HCl, 25mM methionine (trace 2); 20mM histidine-HCl (trace 3). 50 μ L was injected.

FIGS. 14A and 14B show evaluation of A16/A17 protein without PS20 in the mobile phase using 0.15-1.50% ammonium hydroxide additive. FIG. 14A shows an ELSD chromatogram. FIG. 14B shows a UV (280 nm) chromatogram. 50 μ L of A16/A17 protein without PS20 (traces 1,2,3 and 4, respectively) and A16/A17 formulation buffer without PS20 with 1.5% ammonium hydroxide (trace 5) were injected in the mobile phase with 0.15, 0.29, 0.73 and 1.5% ammonium hydroxide.

Figures 15A and 15B show the evaluation of 20-60% mobile phase B wash steps. FIG. 15A shows an ELSD chromatogram. FIG. 15B shows a UV (280 nm) chromatogram. 15 μ L of A16/A17 protein without PS20 was injected. 20,30,40,50 and 60% mobile phase B (wash step), shown as traces 1,2,3,4 and 5, respectively.

FIG. 16 shows the wash time and injection volume of the A16/A17 protein without PS20 assessed by ELSD chromatography. Injection of 25. Mu.L A16/A17 sample without PS20 3.4 min wash step (trace 1). Injection of 50 μ L of A16/A17 sample without PS20, 3.4 min wash step (trace 2) and 2.4 min wash step (trace 3).

FIGS. 17A and 17B show the evaluation of different flow rates for A18/A19 without PS 20. FIG. 17A shows an ELSD chromatogram. FIG. 17B shows a UV (280 nm) chromatogram. 25 μ L of A18/A19 without PS20 (150 mg/mL) were injected with different flow rates of 1.6,1.4,1.25,1.0 and 0.8 mL/min, corresponding to traces 1,2,3,4 and 5, respectively.

Fig. 18 shows the evaluation of different elution times of PS20 in water by ELSD chromatography. 50 μ L was injected with 0.1mg/mL PS20 in water, 3.1 min elution step (trace 1) or 1.1 min elution step (trace 2).

Fig. 19 shows the evaluation of the finalized method 2 by ELSD chromatography. 25 μ L of water for injection (trace 1), 150mg/mL of A18/A19 without PS20 (trace 2), 0.2mg/mL PS20 with water (trace 3) and 0.2mg/mL PS20 with A18/A19 (trace 4), using the finalized parameters of method 2.

FIGS. 20A-20F show the evaluation of specificity for three different products. FIGS. 20A (A18/A19), 20C (A16/A17) and 20E (A14/A20) show ELSD chromatograms. FIGS. 20B (A18/A19), 20D (A16/A17) and 20F (A14/A20) show UV (280 nm) chromatograms. Formulation without PS20 (trace 1), protein without PS20 (trace 2) and 0.1mg/mL PS20 in water (trace 3).

FIG. 21 shows the evaluation of column-to-column variability by ELSD chromatography using PS20 incorporated in water or A18/A19 without PS 20. Cartridge 2, spiked with 0.1mg/mL PS20 of a18/a19 without PS20 (trace 1); cartridge 6, doped with 0.1mg/mL PS20 of a18/a19 without PS20 (trace 2); cartridge 2, 0.2mg/mL PS20 spiked into water (trace 3); and cartridge 6, 0.2mg/mL PS20 in water (trace 4).

FIG. 22 shows the evaluation of column-to-column variability by ELSD chromatography using PS20 incorporated in water or A18/A19 without PS 20. Cartridge 4, spiked with 0.2mg/mL PS20 of a18/a19 without PS20 (trace 1); cartridge 6, doped with 0.2mg/mL PS20 of a18/a19 without PS20 (trace 2); cartridge 4, 0.2mg/mL PS20 spiked in water (trace 3); and cartridge 6, 0.2mg/mL PS20 spiked into water (trace 4).

FIG. 23 shows an ELSD chromatogram of a control sample used in a sequence including 100A 18/A19 injections on a single cartridge. Superposition of 11 control samples injected throughout the sequence.

FIG. 24 shows an ELSD chromatogram of an A18/A19 sample used in a sequence comprising 100A 18/A19 injections on a single cartridge. 100 injections of A18/A19 samples were superimposed throughout the sequence.

FIGS. 25A and 25B show chromatograms of the 1 st, 50 th and 100 th protein injections from a sequence comprising 100A 18/A19 injections on a single column.

FIGS. 26A and 26B show the quantification of 100 injections of A18/A19 samples (nominally 0.2mg/mL PS 20) using cartridge 4. Fig. 26A shows PS20 area versus number of injections. Fig. 26B shows PS20 concentration versus number of injections.

FIGS. 27A and 27B show the quantification of 100 injections of A18/A19 formulation buffer (nominally 0.2mg/mL PS 20) using column 5. Fig. 27A shows PS20 area versus number of injections. Fig. 27B shows PS20 concentration versus number of injections.

FIGS. 28A and 28B show the quantification of 100 injections spiked with 0.2mg/mL PS20 in water using cartridge 3. Fig. 28A shows PS20 area versus number of injections. Fig. 28B shows PS20 concentration versus number of injections.

FIGS. 29A-29F show the evaluation of three low pI products by ELSD chromatography using methods 1 and 2. FIGS. 29A,29C, and 29E show the chromatograms of method 1, using A21, A14/A15, and A14, respectively. FIGS. 29B,29D, and 29F show the chromatograms of method 2, using A21, A14/A15, and A14, respectively.

Detailed description of the invention

The present invention provides methods for quantifying a nonionic surfactant in a composition comprising a polypeptide and a nonionic surfactant, wherein the quantification exhibits reduced interference between the nonionic surfactant and the polypeptide. Methods are also provided wherein the composition further comprises N-acetyltryptophan and quantitatively exhibits reduced interference between the nonionic surfactant, the polypeptide, and the N-acetyltryptophan.

I. Definition of

The terms "polypeptide" or "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers, either naturally or modified by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation to a labeling component or toxin. Also included in the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. The terms "polypeptide" and "protein" as used herein specifically include antibodies.

By "purified" polypeptide (e.g., an antibody or immunoadhesin) is meant that the purity of the polypeptide is increased such that it is present in a more pure form than it is in its natural environment and/or when initially synthesized and/or amplified under laboratory conditions. Purity is a relative term and does not necessarily imply absolute purity.

The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits or neutralizes a biological activity of a native polypeptide. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics the biological activity of a native polypeptide. Suitable agonist or antagonist molecules include in particular agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of the native polypeptide, etc. Methods for identifying agonists or antagonists of a polypeptide can include contacting the polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide.

A polypeptide that "binds" an antigen of interest, e.g., a tumor-associated polypeptide antigen target, is one that binds the antigen with sufficient affinity such that the polypeptide can be used as a diagnostic and/or therapeutic agent that targets cells or tissues that express the antigen and does not significantly cross-react with other polypeptides. In such embodiments, the extent of binding of a polypeptide to a "non-target" polypeptide will be less than about 10% of the binding of the polypeptide to its particular target polypeptide, as determined by Fluorescence Activated Cell Sorting (FACS) analysis or Radioimmunoprecipitation (RIA).

With respect to binding of a polypeptide to a target molecule, the terms "specific binding" or "specific for" a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from non-specific interactions. For example, specific binding can be measured by determining the binding of a molecule as compared to the binding of a control molecule, which is typically a similarly structured molecule that does not have binding activity. For example, specific binding can be determined by competition with a target, e.g., an excess of a control molecule similar to the unlabeled target. In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target.

The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein.

Antibodies are naturally occurring immunoglobulin molecules with different structures, all of which are based on immunoglobulin folding. For example, igG antibodies have two "heavy" chains and two "light" chains that are joined by disulfide bonds to form a functional antibody. Each heavy and light chain itself comprises "constant" (C) and "variable" (V) regions. The V region determines the antigen binding specificity of the antibody, while the C region provides structural support and plays a role in non-antigen specific interactions with immune effectors. The antigen binding specificity of an antibody or antigen binding fragment of an antibody is the ability of the antibody to specifically bind to a particular antigen.

The antigen binding specificity of an antibody is determined by the structural features of the V region. Variability is unevenly distributed over the 110 amino acid span of the variable domain. In contrast, the V regions consist of relatively invariant segments of 15-30 amino acids called Framework Regions (FR) separated by extremely variable, shorter regions called "hypervariable regions" (HVRs) that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, predominantly in a β -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β -sheet structure. The hypervariable regions in each chain are held tightly together by the FRs and together with the hypervariable regions from the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, sequences of Proteins of Immunological Interest,5th Ed. Public Health service, national Institutes of Health, bethesda, md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

Each V region typically comprises three HVRs, e.g., three complementarity determining regions ("CDRs," each comprising a "hypervariable loop") and four framework regions. Thus, an antibody binding site, i.e., the smallest building block required to bind a particular desired antigen with substantial affinity, will generally comprise three CDRs and at least three, and preferably four, framework regions interspersed therebetween in order to maintain and present the CDRs in a suitable conformation. The classical four-chain antibody has an antigen binding site consisting of V _H And V _L Domain collaborative definition. Certain antibodies, such as camel and shark antibodies, lack a light chain and rely solely on a binding site formed by a heavy chain. Single domain engineered immunoglobulins can be prepared in which the binding site is formed only by the heavy or light chain, at V _H And V _L There is no cooperation between them.

The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence between antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed among the variable domains of the antibody. It is concentrated in three segments called hypervariable regions in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FRs, predominantly in a β -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β -sheet structure. The hypervariable regions in each chain are held tightly together by the FRs and together with the hypervariable regions from the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, sequences of Proteins of Immunological Interest,5th Ed. Public Health service, national Institutes of Health, bethesda, md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit a variety of effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

As used herein, the term "hypervariable region" (HVR) refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region may comprise amino acid residues from a "complementarity determining region" or "CDR" (e.g., at V) _L About residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) and at V _H About 31-35B (H1), 50-65 (H2) and 95-102 (H3) (Kabat et al, sequences of Proteins of Immunological Interest,5th Ed. Public Health service, national Institutes of Health, bethesda, md. (1991)) and/or those residues from "high-variable loops" (e.g., V.sub.V.sub.1) _L Residues 26-32 (L1), 50-52 (L2) and 91-96 (L3), and V _H 26-32 (H1), 52A-55 (H2) and 96-101 (H3) (Chothia and Lesk J.mol.biol.196:901-917 (1987)).

"framework" or "FR" residues are those variable domain residues other than the hypervariable region residues defined herein.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, fab ', F (ab') ₂ And Fv fragments; a diabody; tandem diabodies (taDb), linear antibodies (e.g., U.S. Pat. No. 5,641,870, example 2, zapata et al, protein Eng.8 (10): 1057-1062 (1995)); single-arm antibodies, single variable domain antibodies, minibodies, single chain antibody molecules; by antibody fragments (e.g., including, but not limited to, db-Fc, taDb-CH3, (scFV) 4-Fc, di-scFv, bi-scFv, or tandem (di, tri) -scFv); and bispecific T cell adaptors (BiTE)).

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily.Pepsin treatment to yield F (ab') ₂ A fragment having two antigen binding sites and still being capable of cross-linking antigens.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to form a hypervariable region at V _H -V _L The surface of the dimer defines the antigen binding site. The six hypervariable regions together confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the name for Fab' herein, in which the cysteine residues of the constant domains carry at least one free thiol group. F (ab') ₂ Antibody fragments were originally produced as Fab' fragment pairs with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The "light chain" of an antibody (immunoglobulin) from any vertebrate species can be assigned to one of two distinctly different classes, termed kappa and lambda, based on the amino acid sequence of its constant domain.

Antibodies can be assigned to different classes based on the amino acid sequence of their heavy chain constant domains. There are five major classes of intact antibodies, igA, igD, igE, igG and IgM, and some of these may be further divided into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

"Single chain Fv" or "scFv" antibody fragments comprise antibodiesV of _H And V _L Domains, wherein the domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a V _H And V _L A polypeptide linker between the domains that enables the scFv to form the desired structure for antigen binding. For reviews on scFv see Pl ü ckthun in The Pharmacology of Monoclonal Antibodies, vol.113, rosenburg and Moore eds., springer-Verlag, new York, pp.269-315 (1994).

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a light chain variable domain (V) in the same polypeptide chain _L ) Linked heavy chain variable domains (V) _H ) (V _H -V _L ). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al, proc.natl.acad.sci.usa, 90.

The term "multispecific antibody" is used in the broadest sense and specifically covers antibodies having polyepitopic specificity. Such multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (V) _H ) And a light chain variable domain (V) _L ) The antibody of (1), wherein V _H V _L The unit has multi-epitope specificity; having two or more V _L And V _H Antibodies of the Domain, each V _H V _L The units bind different epitopes; an antibody having two or more single variable domains, each single variable domain binding a different epitope; a full-length antibody; antibody fragments such as Fab, fv, dsFv, scFv; a diabody; a bispecific diabody; a triabody; a trifunctional antibody; a covalently or non-covalently linked antibody fragment. "polyepitopic specificity" refers to the ability to specifically bind two or more different epitopes on the same or different targets. By "monospecific" is meant the ability to bind only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that is raised at 5 μ M to 0.001pm,3 μ M to 0.001pm,1 μ M to 0.001pM, 0.5 μ M to 0.001pM, or 0.1 μ M to 0.001pm An affinity of 0.001pM bound to each epitope.

The expression "single domain antibodies" (sdAbs) or "Single Variable Domain (SVD) antibodies" generally refers to antibodies in which a single variable domain (V) is present _H Or V _L ) An antibody that can confer antigen binding. In other words, a single variable domain need not interact with another variable domain to recognize a target antigen. Examples of single domain antibodies include those from camelids (llamas and camels) and cartilaginous fish (e.g. nurse sharks) and those from recombinant methods of human and mouse antibodies (Nature (1989) 341-544 to dev Comp immunocel (2006) 30.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprised in the population are identical and/or bind the same epitope, with these variants generally being present in minor amounts, except for possible variants that may occur during production of the monoclonal antibody. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies also have the advantage that they are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use according to the methods provided herein can be prepared by the hybridoma method first described by Kohler et al, nature 256. "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described in Clackson et al, nature 352, 624-628 (1991) and Marks et al, J.mol.biol.222:581-597 (1991).

Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. nos. 4,816,567 morrison et al, proc. Natl. Acad. Sci. Usa 81. Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (e.g., an old world monkey, such as a baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences (U.S. Pat. No. 5,693,780).

A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal sequences from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity. In some cases, framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications were made to further improve antibody performance. Typically, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence, except for FR substitutions as described above. The humanized antibody optionally will further comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. For further details, see Jones et al, nature 321-525 (1986); riechmann et al, nature 332; and Presta, curr, op, structure, biol.2:593-596 (1992).

For purposes herein, a "whole antibody" is an antibody comprising heavy and light variable domains and an Fc region. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. Preferably, the whole antibody has one or more effector functions.

"native antibodies" are typically heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain at one end (V) _H ) Followed by a number of constant domains. Each light chain has a variable domain at one end (V) _L ) And a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that particular amino acid residues form an interface between the light and heavy chain variable domains.

A "naked antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule, e.g., a cytotoxic moiety or a radiolabel.

In some embodiments, antibody "effector functions" refer to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variable Fc region) and vary with antibody isotype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors.

"antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (fcrs) (e.g., natural Killer (NK) cells, neutrophils, and macrophages) recognize antibody bound on a target cell, followed by lysis of the target cell. Primary cell NK cells mediating ADCC express Fc γ RIII only, whereas monocytes express Fc γ RI, fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is summarized in table 3 on page 464 of ravech and Kinet, annu. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as the assay described in U.S. Pat. No. 5,500,362 or 5,821,337. Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of a molecule of interest can be assessed in vivo, for example in animal models disclosed in Clynes et al, proc.natl.acad.sci. (USA) 95.

"human effector cells" are leukocytes which express one or more fcrs and perform effector functions. In some embodiments, the cells express at least Fc γ RIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; PBMC and NK cells are preferred.

"complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1 q) to a molecule, such as a polypeptide (e.g., an antibody), that complexes with a homologous antigen. To assess complement activation, CDC assays may be performed, for example, as described by Gazzano-Santoro et al, j.immunol.methods 202 (1996).

The term "Fc receptor" or "FcR" is used to describe a receptor that binds the Fc region of an antibody. In some embodiments, the FcR is a native sequence human FcR. In addition, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc γ RI, fc γ RII, and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor Fc γ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see e.g. ibi et al

Annu.Rev.Immunol.15:203-234 (1997)). FcR is found in ravech and Kinet, annu. Rev. Immul 9; capel et al, immunolmethods 4 (1994); and de Haas et al, J.Lab.Clin.Med.126:330-41 (1995). The term "FcR" herein encompasses other fcrs, including those identified in the future. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, j.immunol.117:587 (1976) and Kim et al, j.immunol.24.

"impurities" refers to substances that are different from the desired polypeptide product. In some embodiments of the invention, the impurity comprises a charge variant of the polypeptide. In some embodiments of the invention, the impurity comprises a charge variant of the antibody or antibody fragment. In other embodiments of the invention, impurities include, but are not limited to, host cell material, such as CHOP; leaching out the protein A; a nucleic acid; a variant, fragment, aggregate or derivative of the desired polypeptide; another polypeptide; an endotoxin; viral contaminants; cell culture media components, and the like.

As used herein, the term "immunoadhesin" refers to antibody-like molecules that bind the binding specificity of a heterologous polypeptide to the effector functions of an immunoglobulin constant domain. Structurally, immunoadhesins comprise a fusion of an amino acid sequence having the desired binding specificity, which is distinct from the antigen recognition and binding site of the antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule is generally a contiguous amino acid sequence comprising at least a binding site for a receptor or a ligand. The immunoglobulin constant domain sequence in immunoadhesins can be obtained from any immunoglobulin, for example, igG-1, igG-2, igG-3 or IgG-4 subtypes, igA (including IgA-1 and IgA-2), igE, igD or IgM.

As used herein, "surfactant" refers to a surfactant, preferably a nonionic surfactant. Examples of surfactants herein include polysorbates (e.g., polysorbate 20 and polysorbate 80); poloxamers (e.g., poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; dodecyl-, myristoyl-, linoleyl-or stearyl-sulphoneA betaine; dodecyl-, myristoyl-, linoleyl-or stearyl-sarcosine; linoleyl-, myristyl-or hexadecyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitoamidopropyl-or isostearamidopropyl-betaine (e.g. lauramidopropyl); myristamidopropyl-, palmitoamidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl, or disodium methyl oleyl-taurate; and MONAQUAT ^TM Series (Mona Industries, inc., paterson, n.j.); polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol (e.g., pluronics, PF68, etc.); in one embodiment, the surfactant herein is polysorbate 20. In another embodiment, the surfactant herein is poloxamer 188.

The term "sequential" as used herein with respect to chromatography refers to having a first chromatography followed by a second chromatography. Additional steps may be included between the first chromatography and the second chromatography.

The term "continuous" as used herein with respect to chromatography refers to having a first chromatography material and a second chromatography material directly connected or some other mechanism that allows continuous flow between the two chromatography materials.

"loading density" refers to the amount of composition (e.g., grams) contacted with a volume of chromatography material (e.g., liters). In some examples, the loading density is expressed in g/L.

As used herein, the term "interfere" with respect to the quantification of a species (e.g., a nonionic surfactant) refers to the contribution to the quantification of a certain component (e.g., a polypeptide) other than the species in question. For example, the ELSD signal of a chromatography fraction containing polysorbate 20 and a polypeptide will have contributions from polysorbate 20 and the polypeptide, and quantification of polysorbate 20 in the fraction will have interference from the polypeptide.

As used herein, "substantially the same" means that the value or parameter has not been altered by a significant effect. For example, the ionic strength of the chromatographic mobile phase at the column outlet is substantially the same as the initial ionic strength of the mobile phase if there is no significant change in ionic strength. For example, the ionic strength at the column outlet is substantially the same as the initial ionic strength within 10%, 5%, or 1% of the initial ionic strength.

References herein to a "value or parameter of" about "includes (and describes) variations that are directed to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. It is to be understood that the aspects and variations of the invention described herein include aspects and variations that "consist of and/or" consist essentially of.

Chromatography method

In some aspects, the invention provides methods of analyzing a composition comprising a polypeptide and a non-ionic surfactant (e.g., polysorbate 20 or PS 20), comprising binding the polypeptide and the non-ionic surfactant to a mixed-mode ion-exchange chromatography material using a loading buffer, and eluting the polypeptide and the non-ionic surfactant from the chromatography material using the buffer, such that the polypeptide and the non-ionic surfactant are eluted from the chromatography material in different fractions. In some embodiments, the chromatographic methods are applicable to compositions comprising a plurality of polypeptides (e.g., polypeptide products), including polypeptides having different pis. For example, the method can be used to analyze a composition comprising a non-ionic surfactant and a number of different antibody products (e.g., antibody products with pI ranging from 6.0 to 9.5). In other embodiments, the chromatography method comprises using optimal conditions identified by the methods described herein (e.g., chromatography material, buffer, gradient, step duration, flow rate, sample loading).

In some embodiments of any of the methods described herein, the chromatography material is a mixed mode material comprising functional groups capable of one or more of anion exchange, cation exchange, hydrogen bonding, and hydrophobic interactions. In some embodiments, the mixed mode material is a mixed mode anion exchange chromatography material. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the blend modeThe anion exchange chromatography material comprises quaternary amine moieties. In some embodiments, the mixed mode anion exchange chromatography material is

MAX chromatography material. In some embodiments, the mixed mode material is a mixed mode cation exchange chromatography material. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the mixed mode material comprises a solid support. In some embodiments, the mixed mode material is contained in a column or cartridge. In some embodiments of the foregoing, the mixed mode material is a mixed mode chromatography column or cartridge, such as a mixed mode anion exchange chromatography column or cartridge, or a mixed mode cation exchange chromatography column or cartridge. In some embodiments, the mixed mode material is a High Performance Liquid Chromatography (HPLC) material.

In some embodiments of any of the methods described herein, the ion exchange material can use a conventional chromatography material or a convective chromatography material. Conventional chromatography materials include, for example, perfusion materials (e.g., poly (styrene-divinylbenzene) resins) and diffusion materials (e.g., cross-linked agarose resins). In some embodiments, the poly (styrene-divinylbenzene) resin may be

And (3) resin. In some embodiments, the crosslinked agarose resin may be sulfopropyl-

Fast Flow ("SPSFF") resin. The convective chromatography material can be a membrane (e.g., polyethersulfone) or a monolithic material (e.g., cross-linked polymer). The polyethersulfone membrane canTo be Mustang. The crosslinked polymer monolith material may be crosslinked poly (glycidyl methacrylate-co-ethylene dimethacrylate).

In some embodiments of any of the methods of the invention, the chromatography material is in a chromatography column or cartridge; for example, a mixed mode cation exchange chromatography column or cartridge or a mixed mode anion exchange chromatography column or cartridge. In some embodiments, a chromatography column or cartridge is used for liquid chromatography. In some embodiments, the chromatography column or cartridge is used for High Performance Liquid Chromatography (HPLC). In some embodiments, the chromatography column or cartridge is an HPLC chromatography column or cartridge; for example, a mixed mode cation exchange HPLC column or cartridge or a mixed mode anion exchange HPLC column or cartridge.

For example, in some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of the acid; b) Eluting the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the nonionic surfactant in the eluate of step c). In some embodiments, the polypeptide specifically and non-specifically binds to a chromatography material, and at least about 90% (e.g., at least any one of about 91, 92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01%, or less) of total polypeptides in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 30. In some embodiments, the third ratio is between about 80 to about 100 (e.g., about 82. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) of an acid (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises from about 0.5% to about 5% (v/v) (e.g., any of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) of an acid in methanol. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid.

In some embodiments, the flow rate of the chromatography is about 0.5 to 2.5 (e.g., about any one of 0.7,0.9,1.1, 1.2,1.25,1.3,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatography material is about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20, 25,30,35,40, and 45, including any range between these values) μ Ι _.

In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9, 2, or more) minutes after the beginning of step a) and continues for at least about 1 (e.g., at least about any one of 1.2,1.4, 1.6,1.8,2,2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8,4 or more) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least any one of about 0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5, 1,1.5,2, or more) minutes after the end of step b) and continues for at least about 0.5 (e.g., at least about any of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,2,3, or more) minutes. In some embodiments, the nonionic surfactant is a poloxamer (P188) or a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the nonionic surfactant in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01,0.05,0.1,0.2,0.3, 0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of protein in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4, 5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, thiomab ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises quaternary amine moieties. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is

MAX chromatography materials. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%, 0.01%, or lower) polypeptide interference.

In some embodiments, a method for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of the acid; b) Eluting the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the nonionic surfactant in the eluate of step c), wherein the quantifying of the nonionic surfactant comprises less than about 10% (e.g., any of less than about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%, 0.01%, or less) interference from the polypeptide. In some embodiments, the polypeptide binds specifically and non-specifically to the chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%, 0.01% or less) of total polypeptides in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0 Between 0 and about 20 (e.g., about any one of 2. In some embodiments, the second ratio is between about 30. In some embodiments, the third ratio is between about 80. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) acid (e.g., any one of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises from about 0.5% to about 5% (v/v) (e.g., any of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) of an acid in methanol. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.2,1.25,1.3,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatography material is about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7, 8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ Ι _. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5, 1.6,1.7,1.8,1.9,2, or more) minutes after the beginning of step a) and continues for at least about 1 (e.g., at least about any of 1.2,1.4,1.6,1.8,2,2.2,2.4,2.6,2.8,3,3.2,3.4, 3.6,3.8,4 or more) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least any one of about 0.06,0.07,0.08,0.09,0.1, 0.2,0.3,0.4,0.5,1,1.5,2 or more) minutes after the end of step b) and continues for at least about 0.5 (e.g., any one of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,2, 3 or more) minutes. In some embodiments, the nonionic The surfactant is poloxamer (P188) or polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the nonionic surfactant in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01,0.05, 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of protein in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120,140, 160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6, 4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, thiomab ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is

MAX chromatography material. In some embodiments, the light is evaporatedScattering (ELSD) or by using a Charged Aerosol Detector (CAD) to quantify non-ionic detergents.

In some embodiments, a method for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of the acid; b) Eluting the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the nonionic surfactant in the eluate of step c), wherein the eluate comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%, 0.01% or less) of the total polypeptides in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 30. In some embodiments, the third ratio is between about 80. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) of an acid (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises about 0.5% to about 5% (v/v) (e.g., about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4% Any of 5%, including any range between these values) acid in methanol. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.2,1.25,1.3,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7, 8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5, 1.6,1.7,1.8,1.9,2, or more) minutes after the beginning of step a) and continues for at least about 1 (e.g., at least about any of 1.2,1.4,1.6,1.8,2,2.2,2.4,2.6,2.8,3,3.2,3.4, 3.6,3.8,4 or more) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least any one of about 0.06,0.07,0.08,0.09,0.1, 0.2,0.3,0.4,0.5,1,1.5,2, or more) minutes and continues for at least about 0.5 (e.g., at least any one of about 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5, 2,3, or more) minutes after the end of step b). In some embodiments, the nonionic surfactant is a poloxamer (P188) or a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the nonionic surfactant in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01, 0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of protein in the composition is from about 1mg/mL to about 250mg/mL (e.g., any of about 2,5,10,20,40,60,80,100,120, 140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0, 7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more agents selected from the group consisting of stabilizers, buffers and tonicity agents Excipients for the agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises quaternary amine moieties. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is

MAX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%, 2%,1%,0.5%,0.1%,0.05%,0.01%, or lower) interference from the polypeptide.

In some embodiments, a method for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous solution of acetic acid and mobile phase B comprises a methanol solution of acetic acid; b) From mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase AEluting the polypeptides, wherein the second ratio is greater than the first ratio; c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the non-ionic surfactant in the eluate of step c). In some embodiments, the polypeptide specifically and non-specifically binds to a chromatography material, and at least about 90% (e.g., at least any one of about 91, 92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) of the total polypeptides in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 30. In some embodiments, the third ratio is between about 80, 20 to about 100 (e.g., about 82. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) acetic acid (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises a methanol solution of acetic acid from about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.2,1.25,1.3,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the application is to a chromatographic material The composition of (a) is about 1 to about 50 (e.g., about any of 1,2,3,4,5, 6,7,8,9,10,15,20,25,30,35,40, and 45, including any range therebetween) μ L in volume. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5, 1.6,1.7,1.8,1.9,2, or more) minutes after the beginning of step a) and continues for at least about 1 (e.g., at least about any of 1.2,1.4,1.6,1.8,2,2.2,2.4,2.6,2.8,3,3.2, 3.4,3.6,3.8,4, or more) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any of 0.06,0.07,0.08,0.09, 0.1,0.2,0.3,0.4,0.5,1,1.5,2, or more) minutes and continues for at least about 0.5 (e.g., any of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5, 2,3, or more) minutes after the end of step b). In some embodiments, the nonionic surfactant is a poloxamer (P188) or a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the nonionic surfactant in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01, 0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of protein in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120, 140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0, 7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, THIOMAB ^TM Or THIOMAB ^TM A drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises quaternary amine moieties. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is

MAX chromatography materials. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%, 2%,1%,0.5%,0.1%,0.05%,0.01%, or lower) polypeptide interference.

In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a mobile phase B and a mobile phase a in a first ratio of between about 5; b) Eluting the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a between about 35; c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a of between about 90; d) Quantifying the nonionic surfactant in the eluate of step c). In some embodiments, the first ratio of mobile phase B to mobile phase a is about 10. In some embodiments, the second ratio is about 40: 60. In some embodiments, the third ratio is about 100. In some embodiments, the polypeptide specifically and non-specifically binds to a chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%, 0.1%,0.05%,0.01% or less) of the total polypeptides in the composition. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) of an acid (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises a methanol solution of about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) acid. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.2,1.25,1.3,1.5,1.7,1.9, 2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2, 3,4,5,6,7,8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4, 1.5,1.6,1.7,1.8,1.9,2, or more) minutes after the beginning of step a) and continues for at least about 1 (e.g., at least about any of 1.2,1.4,1.6,1.8,2,2.2,2.4,2.6,2.8,3, 3.2,3.4,3.6,3.8,4 or more) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one of 0.06,0.07,0.08, 0.09,0.1,0.2,0.3,0.4,0.5,1,1.5,2 or more) minutes after the end of step b) and continues for at least about 0.5 (e.g., any one of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4, 1.5,2,3 or more) minutes . In some embodiments, the nonionic surfactant is a poloxamer (P188) or a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the nonionic surfactant in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005, 0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of protein in the composition is from about 1mg/mL to about 250mg/mL (e.g., any of about 2,5,10,20,40,60,80,100, 120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0, 7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is

MAX chromatography material. In thatIn some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%, 2%,1%,0.5%,0.1%,0.05%,0.01%, or lower) polypeptide interference.

In some embodiments, a method for quantifying polysorbate in a composition comprising polysorbate and polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of the acid; b) Eluting the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the polysorbate in the eluate of step c). In some embodiments, the polypeptide binds specifically and non-specifically to the chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%, 2%,1%,0.5%,0.1%,0.05%,0.01% or less) of the total polypeptides in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 30 Surround). In some embodiments, the third ratio is between about 80. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) acid (e.g., any one of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises a methanol solution of about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) acid. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.2,1.25,1.3,1.5,1.7,1.9, 2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2, 3,4,5,6,7,8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4, 1.5,1.6,1.7,1.8,1.9,2, or more) minutes after the beginning of step a) and continues for at least about 1 (e.g., at least about any of 1.2,1.4,1.6,1.8,2,2.2,2.4,2.6,2.8,3, 3.2,3.4,3.6,3.8,4, or more) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any of 0.06,0.07,0.08, 0.09,0.1,0.2,0.3,0.4,0.5,1,1.5,2, or more) minutes and continues for at least about 0.5 (e.g., any of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4, 1.5,2,3, or more) minutes after the end of step b). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01,0.05, 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the protein concentration in the composition is About 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120,140, 160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., any of about 4.6, 4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, thiomab ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is

MAX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01%, or lower) polypeptide interference.

In some embodiments, there is provided a method for quantifying polysorbate in a composition comprising polysorbate and a polypeptideA method of esters, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of the acid; b) Eluting the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the polysorbate in the eluate of step c), wherein the quantifying of the nonionic surfactant comprises less than about 10% (e.g., less than about any one or less of 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%, 0.01%) interference from the polypeptide. In some embodiments, the polypeptide specifically and non-specifically binds to a chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93, 94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) of the total polypeptides in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 30. In some embodiments, the third ratio is between about 80. In some embodiments, mobile phase a comprises about 0.5% to about 5% (v/v) (e.g., about Any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) of an acid. In some embodiments, mobile phase B comprises a methanol solution of about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) acid. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.2, 1.25,1.3,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20,25, 30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any of 0.6,0.7, 0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2, or more) minutes after the beginning of step a) and continues for at least about 1 (e.g., at least about any of 1.2,1.4,1.6, 1.8,2,2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8,4 or more) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any of 0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,1, 1.5,2, or more) minutes and continues for at least about 0.5 (e.g., any of 0.6,0.7, 0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,2,3, or more) minutes after the end of step b). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6, 0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of protein in the composition is from about 1mg/mL to about 250mg/mL (e.g., any of about 2,5, 10,20,40,60,80,100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0, 6.2,6.4,6.6,6.8,7.0,7 Any of 2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, thiomab ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is

MAX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD).

In some embodiments, a method is provided for quantifying polysorbate in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of the acid; b) Eluting the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) By including a third ratioThe solution of mobile phase B and mobile phase a of example elutes polysorbate from the chromatography material, wherein the third ratio is greater than the second ratio; d) Quantifying polysorbate in the eluate of step c), wherein the eluate comprises less than about 10% (e.g., less than any of about 9%,8%,7%, 6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) of total polypeptide in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 30. In some embodiments, the third ratio is between about 80. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) acid (e.g., any one of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises a methanol solution of about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) acid. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.2, 1.25,1.3,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20,25, 30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any of 0.6, 0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2, or more) minutes after the beginning of step a) and continues until step a) begins About 1 (e.g., at least about any of 1.2,1.4,1.6, 1.8,2,2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8,4, or more) minutes less. In some embodiments, step c) begins at least about 0.05 (e.g., at least any one of about 0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,1, 1.5,2, or more) minutes after the end of step b) and continues for at least about 0.5 (e.g., any one of 0.6,0.7, 0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,2,3, or more) minutes. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6, 0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of protein in the composition is from about 1mg/mL to about 250mg/mL (e.g., any of about 2,5, 10,20,40,60,80,100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., any of about 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0, 6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, thiomab ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises quaternary amine moieties. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, mixingThe model anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is

MAX chromatography materials. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01%, or lower) polypeptide interference.

In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate and polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous solution of acetic acid and mobile phase B comprises a methanol solution of acetic acid; b) Eluting the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the polysorbate in the eluate of step c). In some embodiments, the polypeptide specifically and non-specifically binds to a chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01%, or less) of total polypeptides in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0 6, 94, 92, 10. In some embodiments, the second ratio is between about 30. In some embodiments, the third ratio is between about 80. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) acetic acid (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises a methanol solution of acetic acid at about 0.5% to about 5% (v/v) (e.g., any of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.2,1.25, 1.3,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35, 40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any of 0.6,0.7,0.8, 0.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2, or more) minutes after the beginning of step a) and continues for at least about 1 (e.g., at least about any of 1.2,1.4,1.6,1.8, 2,2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8,4 or more) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any of 0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,1,1.5, 2, or more) minutes and continues for at least about 0.5 (e.g., any of 0.6,0.7,0.8, 0.9,1,1.1,1.2,1.3,1.4,1.5,2,3, or more) minutes after the end of step b). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is at In the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005,0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7, 0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of protein in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10, 20,40,60,80,100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0, 6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, thiomab ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises quaternary amine moieties. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is

MAX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the basis weight of the nonionic surfactant comprises less than about 10% (e.g., less than about 9%,8%,7%,6%,5%,4%, 3%)2%,1%,0.5%,0.1%,0.05%,0.01%, or lower).

In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate and polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a of between about 5 to about 15, wherein mobile phase a comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of the acid; b) Eluting the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a between about 35; c) Eluting the polysorbate from the chromatography material with a solution comprising mobile phase B to mobile phase a in a third ratio of between about 90; d) Quantifying the polysorbate in the eluate of step c). In some embodiments, the first ratio of mobile phase B to mobile phase a is about 10. In some embodiments, the second ratio is about 40. In some embodiments, the third ratio is about 100. In some embodiments, the polypeptide specifically and non-specifically binds to a chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) of the total polypeptides in the composition. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) of an acid (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises a methanol solution of about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) acid. In some embodiments, the acid is Formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.2,1.25,1.3,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5, 6,7,8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5, 1.6,1.7,1.8,1.9,2, or more) minutes after the beginning of step a) and continues for at least about 1 (e.g., at least about any of 1.2,1.4,1.6,1.8,2,2.2,2.4,2.6,2.8,3,3.2, 3.4,3.6,3.8,4 or more) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least any one of at least about 0.06,0.07,0.08,0.09, 0.1,0.2,0.3,0.4,0.5,1,1.5,2 or more) minutes after the end of step b) and continues for at least about 0.5 (e.g., any one of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5, 2,3 or more) minutes. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005,0.01,0.05,0.1, 0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of protein in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120,140,160, 180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0, 5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, polyclonal Antibodies, monoclonal antibodies, humanized antibodies, human antibodies, chimeric antibodies, multispecific antibodies, glycoengineered antibodies, antibody fragments, antibody drug conjugates, thiomab ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises quaternary amine moieties. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is

MAX chromatography materials. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%, 0.1%,0.05%,0.01%, or lower) polypeptide interference.

In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate and polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a between about 5 to about 15, wherein mobile phase a comprises an aqueous solution of acetic acid and mobile phase B comprises a methanol solution of acetic acid; b) Eluting the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a between about 35; c) Eluting the polysorbate from the chromatography material with a solution comprising mobile phase B to mobile phase a in a third ratio of between about 90; d) Quantifying the eluate of step c)The polysorbate of (4), wherein the quantification of the nonionic surfactant includes less than about 10% (e.g., less than about any one or less of 9%,8%,7%,6%,5%,4%,3%,2%,1%, 0.5%,0.1%,0.05%, 0.01%) interference from the polypeptide. In some embodiments, the first ratio of mobile phase B to mobile phase a is about 10. In some embodiments, the second ratio is about 40. In some embodiments, the third ratio is about 100. In some embodiments, the polypeptide specifically and non-specifically binds to a chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%, 0.5%,0.1%,0.05%,0.01% or less) of the total polypeptides in the composition. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) acetic acid (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises a methanol solution of acetic acid from about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.2, 1.25,1.3,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatography material is about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20,25, 30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any of 0.6, 0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2, or more) minutes after the beginning of step a) and continues for at least about 1 (e.g., at least about any of 1.2,1.4,1.6, 1.8,2,2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8,4, or more) minutes. In that In some embodiments, step c) begins at least about 0.05 (e.g., at least any one of about 0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,1, 1.5,2, or more) minutes after the end of step b) and continues for at least about 0.5 (e.g., any one of 0.6,0.7, 0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,2,3, or more) minutes. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6, 0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of protein in the composition is from about 1mg/mL to about 250mg/mL (e.g., any of about 2,5, 10,20,40,60,80,100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., any of about 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0, 6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises quaternary amine moieties. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, mixed mode anionsThe exchange chromatography material is

MAX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD).

In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate and polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a between about 5 to about 15, wherein mobile phase a comprises an aqueous solution of acetic acid and mobile phase B comprises a methanol solution of acetic acid; b) Eluting the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a between about 35; c) Eluting the polysorbate from the chromatography material with a solution comprising mobile phase B to mobile phase a in a third ratio of between about 90; d) Quantifying polysorbate in the eluate of step c), wherein the eluate from step c) comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%, 1%,0.5%,0.1%,0.05%,0.01% or less) of total polypeptide in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is about 10. In some embodiments, the second ratio is about 40. In some embodiments, the third ratio is about 100. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) acetic acid (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises a methanol solution of acetic acid from about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about 0.7,0.9, 1.1,1.2,1.25,1.3,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatography material is about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15, 20,25,30,35,40, and 45, including any range between these values) μ Ι _. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8, 1.9,2, or more) minutes after the beginning of step a) and continues for at least about 1 (e.g., at least any one of about 1.2, 1.4,1.6,1.8,2,2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8,4 or more) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least any one of about 0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4, 0.5,1,1.5,2, or more) minutes after the end of step b) and continues for at least about 0.5 (e.g., at least any one of about 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,2,3, or more) minutes. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005,0.01,0.05,0.1,0.2,0.3, 0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of protein in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., any of about 4.6,4.8,5.0,5.2,5.4, 5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises quaternary amine moieties. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is

MAX chromatography materials. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01%, or lower) interference from the polypeptide.

In some embodiments, a method is provided for quantifying polysorbate 20 in a composition comprising polysorbate and polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a ratio of about 10 mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous solution of about 2% acetic acid and mobile phase B comprises a methanol solution of about 2% acetic acid; b) Eluting the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising mobile phase B and mobile phase a in a ratio of about 40; c) Eluting polysorbate 20 from the chromatography material with a solution comprising mobile phase B to mobile phase a in a ratio of about 100; d) Quantifying polysorbate 20 in the eluate of step c), in some embodiments the eluate from step c) comprises less than about 10% (e.g., less than about any of 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) One) of the compositions of (a). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.2,1.25,1.3,1.5,1.7,1.9, 2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the flow rate for chromatography is about 1.25mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9, 10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, the volume of the composition applied to the chromatographic material is about 20 μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7, 1.8,1.9,2, or more) minutes after the beginning of step a) and continues for at least about 1 (e.g., at least about any of 1.2,1.4,1.6,1.8,2,2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8, 4, or more) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least any one of about 0.06,0.07,0.08,0.09,0.1,0.2,0.3, 0.4,0.5,1,1.5,2, or more) minutes and continues for at least about 0.5 (e.g., at least any one of about 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,2,3, or more) minutes after the end of step b). In some embodiments, the concentration of polysorbate 20 in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005,0.01,0.05, 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of protein in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120,140, 160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6, 4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some cases In embodiments, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is

MAX chromatography materials. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%, 0.5%,0.1%,0.05%,0.01%, or lower) interference from the polypeptide.

In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the non-ionic table from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase AA surfactant, wherein the third ratio is greater than the second ratio; d) Quantifying the nonionic surfactant in the eluate of step c). In some embodiments, the polypeptide binds specifically and non-specifically to the chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%, 0.01% or less) of the total polypeptides in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of mobile phase B is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 35 to about 55 (e.g., about 36. In some embodiments, the third ratio is between about 80. In some embodiments, mobile phase a comprises an aqueous solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises an organic solvent (e.g., methanol or acetonitrile) solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.3,1.4,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments of the present invention, the substrate is, The volume of the composition applied to the chromatographic material is about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10, 15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7, 1.8,1.9,2, or more) minutes after the beginning of step a) and continues for at least about 2 (e.g., at least about any of 2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8,4,4.5,5, or more) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any of 0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5, 1,1.5,2, or more) minutes after the end of step b) and continues for at least about 2 (e.g., at least about any of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4,3.6,3.8,4,4.5,5, or more) minutes. In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of the nonionic surfactant (e.g., polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the composition further comprises N-acetyl tryptophan (also known as N-acetyl-DL-tryptophan) and/or methionine. In some embodiments, the concentration of N-acetyltryptophan in the composition is from about 0.1mM to about 10mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the concentration of methionine in the composition is in the range of about 0.1mM to about 100mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6,7,8,9, 10,20,30,40,50,60,70,80, or 90mM, including any range therebetween). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., any of about 2,5,10,20,40,60,80,100,120,140,160, 180,200,220, and 240mg/mL, including any range between these values). At one end In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0, 5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01%, or lower) polypeptide interference.

In some embodiments, a method for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto a membrane in a first ratio comprising the nonionic surfactant and the polypeptideAnd a mobile phase a, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the nonionic surfactant in the eluate of step c), wherein the quantifying of the nonionic surfactant comprises less than about 10% (e.g., less than any one of 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) interference from the polypeptide. In some embodiments, the polypeptide specifically and non-specifically binds to a chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) of total polypeptides in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of mobile phase B is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 35, 65 to about 55 (e.g., about 36. In some embodiments, the third ratio is between about 80. In some embodiments, mobile phase a comprises about 0.5% to about 5% (v/v) (e.g., a solution of sodium chloride and sodium chloride in water) Such as any of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises from about 0.5% to about 5% (v/v) (e.g., any of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) of ammonium hydroxide in an organic solvent (e.g., methanol or acetonitrile). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.3,1.4,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatography material is about 1 to about 50 (e.g., about any of 1,2,3, 4,5,6,7,8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ Ι _. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3, 1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8,4, 4.5, 5) minutes after step a) begins. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08,0.09,0.1,0.2, 0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., any one or more of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4,3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of the nonionic surfactant (e.g., polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6, 0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the composition further comprises N-acetyltryptophan and/or methionine. In some embodiments, the N-acetyl group in the composition The concentration of tryptophan ranges from about 0.1mM to about 10mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8,9,10,20,30,40, 50,60,70,80, or 90mM, including any range between these values). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4, 5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, detection is by Evaporative Light Scattering (ELSD) or by using charged aerosolA machine (CAD) to quantify the nonionic detergent.

In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the non-ionic surfactant in the eluate of step c), wherein the eluate from step c) comprises less than about 10% (e.g., less than about any one of 9%,8%,7%,6%,5%,4%,3%,2%, 1%,0.5%,0.1%,0.05%,0.01% or less) of the total polypeptide in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of mobile phase B is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 35 to about 55 (e.g., about 36. In some embodiments, the third ratio is between about 80 to about 100 (e.g., about 82. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) (e.g., any one of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) ammonium hydroxide. At one end In some embodiments, mobile phase B comprises an organic solvent (e.g., methanol or acetonitrile) solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%, 2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.3,1.4,1.5,1.7,1.9, 2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatography material is about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8, 9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3, 1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8,4, 4.5, 5) minutes after step a) begins. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08,0.09,0.1,0.2, 0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., any one or more of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4,3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of the nonionic surfactant (e.g., polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6, 0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the composition further comprises N-acetyltryptophan and/or methionine. In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., about any of 0.2,0.5, 1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some cases In embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6,7,8,9,10,20,30,40, 50,60,70,80, or 90mM, including any range therebetween). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., any of about 4.6,4.8,5.0,5.2,5.4, 5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the basis weight of the nonionic surfactant comprises less than about 10% (e.g., less than about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01%, or lower).

In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises a methanolic ammonium hydroxide solution; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the non-ionic surfactant in the eluate of step c). In some embodiments, the polypeptide specifically and non-specifically binds to a chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) of total polypeptides in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 35 to about 55 (e.g., about 36. In some embodiments, the third ratio is between about 80 to about 100 (e.g. about any one of 82, 18,84 Any range). In some embodiments, mobile phase a comprises an aqueous solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises a methanolic solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.3,1.4,1.5, 1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1, 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes after the beginning of step a) and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.2,3.4, 3.6,3.8,4,4.5, 5) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08, 0.09,0.1,0.2,0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4, 3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of the nonionic surfactant (e.g., polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01,0.05,0.1,0.2, 0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the composition further comprises N-acetyltryptophan and And/or methionine. In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6,7,8, 9,10,20,30,40,50,60,70,80, or 90mM, including any range between these values). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120, 140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0, 7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodimentsThe nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than any of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01%, or lower) polypeptide interference.

In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises an acetonitrile solution of ammonium hydroxide; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the nonionic surfactant in the eluate of step c). In some embodiments, the polypeptide binds specifically and non-specifically to the chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) of total polypeptides in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 35 Including any range between these ratios). In some embodiments, the third ratio is between about 80 to about 100 (e.g., about 82. In some embodiments, mobile phase a comprises an aqueous solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises from about 0.5% to about 5% (v/v) (e.g., any of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) ammonium hydroxide in acetonitrile. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.3,1.4,1.5, 1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1, 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes after the beginning of step a) and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.2,3.4, 3.6,3.8,4,4.5, 5) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08, 0.09,0.1,0.2,0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4, 3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of the nonionic surfactant (e.g., polysorbate or poloxamer) in the composition is at about In the range of 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01,0.05,0.1,0.2, 0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the composition further comprises N-acetyltryptophan and/or methionine. In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6,7,8, 9,10,20,30,40,50,60,70,80, or 90mM, including any range between these values). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120, 140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0, 7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodimentsThe mixed-mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than about any of 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%, 0.05%,0.01% or less) polypeptide interference.

In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a between about 5 to about 85, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a of between about 40; c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a of between about 90; d) Quantifying the nonionic surfactant in the eluate of step c). In some embodiments, the polypeptide specifically and non-specifically binds to a chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95,96, 97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%, 0.05%,0.01% or less) of total polypeptides in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of mobile phase B is And (3) acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 35 to about 55 (e.g., about 36. In some embodiments, the third ratio is between about 80 to about 100 (e.g., about 82. In some embodiments, mobile phase a comprises an aqueous solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises an organic solvent (e.g., methanol or acetonitrile) solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9, 1.1,1.3,1.4,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20, 25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes after the beginning of step a) and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6, 2.8,3,3.2,3.4,3.6,3.8,4,4.5, 5) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,1,1.5, 2) minutes after the end of step b) and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6, 2.8,3,3.1,3.2,3.4,3.6,3.8,4,4.5, 5) More) minutes. In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of the nonionic surfactant (e.g., polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01, 0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the composition further comprises N-acetyltryptophan and/or methionine. In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8, 9,10,20,30,40,50,60,70,80, or 90mM, including any range between these values). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80, 100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6, 6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises transA phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than any one or less of about 9%,8%,7%,6%,5%,4%,3%, 2%,1%,0.5%,0.1%,0.05%, 0.01%) polypeptide interference.

When the formulation was tested for products containing N-acetyltryptophan (NAT) using HPLC-ELSD conditions, significant interference was observed in the PS20 region. Therefore, in some cases, alternative conditions are needed to eliminate NAT and protein related interference.

In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant, a polypeptide, and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the non-ions in the eluate of step c)A surfactant. In some embodiments, the polypeptide specifically and non-specifically binds to a chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%, 0.1%,0.05%,0.01% or less) of total polypeptides in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (e.g., less than about 4%,3%,2%,1%,0.5%, 0.1%,0.05%,0.01% of any one or less) of total NAT in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of mobile phase B is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 35 to about 55 (e.g., about 36. In some embodiments, the third ratio is between about 80 to about 100 (e.g., about 82. In some embodiments, mobile phase a comprises an aqueous solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises an organic solvent (e.g., methanol or acetonitrile) solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about 0.7,0.9,1.1,1.3,1.4,1.5, 1.7,1.9,2 Any of, 1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatography material is about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1, 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes after the beginning of step a) and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.2,3.4, 3.6,3.8,4,4.5, 5) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08, 0.09,0.1,0.2,0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4, 3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of the nonionic surfactant (e.g., polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01,0.05,0.1,0.2, 0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the composition further comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6,7,8,9,10,20, 30,40,50,60,70,80, or 90mM, including any range therebetween). In some embodiments, the concentration of polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., of about 2,5,10,20,40,60,80,100,120,140,160,180,200,220, and 240mg/mL Any one, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4, 5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the non-ionic surface detergent comprises less than about 10% (e.g., less than about any of 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) polypeptide interference.

In some embodiments, a method for quantifying polysorbate in a composition comprising polysorbate and polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the groupLoading the composition onto a chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the polysorbate in the eluate of step c). In some embodiments, the polypeptide binds specifically and non-specifically to the chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95, 96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) of total polypeptides in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of mobile phase B is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 35. In some embodiments, the third ratio is between about 80 to about 100 (e.g., about 82. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) (e.g., any one of about 0.75%,1%,1.5%,2%,2.5%, 3%,3.5%,4%, and 4.5%, including any range between these values) ammonium hydroxide. In some embodiments of the present invention, the substrate is, Mobile phase B comprises an organic solvent (e.g., methanol or acetonitrile) solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9, 1.1,1.3,1.4,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20, 25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6, 2.8,3,3.2,3.4,3.6,3.8,4,4.5, 5) minutes after step a) begins. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6, 2.8,3,3.1,3.2,3.4,3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate is in the range of about 0.001% to 1.0% (w/v) (e.g., about any of 0.005,0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the composition further comprises N-acetyltryptophan and/or methionine. In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8,9,10,20,30,40,50,60, 70,80, or 90mM, including any range between these values). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5, 10,20,40,60,80,100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., any of about 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0, 6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the polysorbate comprises less than about 10% (e.g., less than about any of 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%, 0.1%,0.05%,0.01% or less) polypeptide interference.

In some embodiments, methods are provided for quantifying polysorbate in a composition comprising polysorbate, polypeptide, and N-acetyltryptophan, wherein the methodThe method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase A, wherein mobile phase A comprises an aqueous ammonium hydroxide solution and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the polysorbate in the eluate of step c), wherein the quantifying of the polysorbate comprises less than about 10% (e.g., less than about any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%, 0.1%,0.05%,0.01% or less) interference from the polypeptide and NAT. In some embodiments, the polypeptide specifically and non-specifically binds to a chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%, 0.1%,0.05%,0.01% or less) of total polypeptides in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (e.g., less than about 4%,3%,2%,1%,0.5%, 0.1%,0.05%,0.01% of any one or less) of total NAT in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of mobile phase B is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 35, 65 to about 55 (e.g., about any one of 36, Including any range between these ratios). In some embodiments, the third ratio is between about 80. In some embodiments, mobile phase a comprises an aqueous solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises an organic solvent (e.g., methanol or acetonitrile) solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.3,1.4,1.5,1.7,1.9, 2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2, 3,4,5,6,7,8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3, 1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8,4, 4.5, 5) minutes after step a) begins. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08,0.09,0.1,0.2, 0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., any one or more of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4,3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005,0.01,0.05,0.1,0.2, 0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from About 0.1mM to about 10mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the composition further comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8,9,10,20, 30,40,50,60,70,80, or 90mM, including any range between these values). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4, 5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the light scattering is by Evaporation (ELSD)) Or by using a Charged Aerosol Detector (CAD) to quantify the non-ionic detergent.

In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate, polypeptide, and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying polysorbate in the eluate of step c), wherein the eluate from step c) comprises less than about 10% (e.g., less than about any one of 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%, 0.1%,0.05%,0.01% or less) of total polypeptides in the composition and less than about 5% (e.g., less than about any one of 4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) of total NAT in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of mobile phase B is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 35 to about 55 (e.g., about 36. In some embodiments, the third ratio is between about 80 to about 100 (e.g., about 82. In some embodiments, mobile phase a comprises an aqueous solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises an organic solvent (e.g., methanol or acetonitrile) solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9, 1.1,1.3,1.4,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20, 25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6, 2.8,3,3.2,3.4,3.6,3.8,4,4.5, 5) minutes after step a) begins. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6, 2.8,3,3.1,3.2,3.4,3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005,0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6, 0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the composition further comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80, or 90mM, including any range between these values). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40, 60,80,100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4, 6.6,6.8,7.0,7.2,7.4, including any range between these values).

In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of polysorbate comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%, 0.5%,0.1%,0.05%,0.01% or less) from more thanInterference of peptides and NAT.

In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate, polypeptide, and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises a methanolic ammonium hydroxide solution; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the polysorbate in the eluate of step c). In some embodiments, the polypeptide binds specifically and non-specifically to the chromatography material, and at least about 90% (e.g., at least any one of about 91,92, 93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%, 0.5%,0.1%,0.05%,0.01% or less) of total polypeptides in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (e.g., less than about 4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% of any one or less) of total NAT in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 35. In some embodiments, the third ratio is between about 80 to about 100 (e.g., about 82, 18, 84. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) (e.g., any one of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) ammonium hydroxide. In some embodiments, mobile phase B comprises a methanolic solution of ammonium hydroxide in the range of about 0.5% to about 5% (v/v) (e.g., any of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.3,1.4,1.5, 1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatography material is about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1, 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.2,3.4, 3.6,3.8,4,4.5, 5) minutes after step a) begins. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08, 0.09,0.1,0.2,0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4, 3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005, 0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., about any of 0.2,0.5,1,2,3,4,5, 6,7,8, or 9mM, including any range between these values). In some embodiments of the present invention, the substrate is, The composition further comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6, 7,8,9,10,20,30,40,50,60,70,80, or 90mM, including any range between these values). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120, 140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0, 7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of polysorbate comprises less than about 10%(e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%, 0.01% or less) interference from the polypeptide and NAT.

In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate, polypeptide, and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises an acetonitrile solution of ammonium hydroxide; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a, wherein the second ratio is greater than the first ratio; c) Eluting the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a, wherein the third ratio is greater than the second ratio; d) Quantifying the polysorbate in the eluate of step c). In some embodiments, the polypeptide binds specifically and non-specifically to the chromatography material, and at least about 90% (e.g., at least any one of about 91,92, 93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%, 0.01% or less) of total polypeptides in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (e.g., less than about 4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% of any one or less) of total NAT in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is between about 0. In some embodiments, the second ratio is between about 35 And including any range between these ratios). In some embodiments, the third ratio is between about 80. In some embodiments, mobile phase a comprises an aqueous solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises from about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) ammonium hydroxide in acetonitrile. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.3,1.4,1.5, 1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1, 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.2,3.4, 3.6,3.8,4,4.5, 5) minutes after step a) begins. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08, 0.09,0.1,0.2,0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., any one or more of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4,3.6, 3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate is in the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005,0.01,0.05, 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., about 0.2,0.5,1 Any of 2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the composition further comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6,7,8,9,10, 20,30,40,50,60,70,80, or 90mM, including any range between these values). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120,140,160,180, 200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., any of about 4.6,4.8,5.0, 5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, by Evaporative Light Scattering (ELSD) or by using charged aerosol detectors(CAD) to quantify the nonionic detergent. In some embodiments, the quantification of polysorbate comprises less than about 10% (e.g., less than about any of 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) interference from the polypeptide and NAT.

In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate, polypeptide, and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a between about 5 to about 15, 85, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a of between about 40; c) Eluting the polysorbate from the chromatography material with a solution comprising mobile phase B to mobile phase a at a third ratio of between about 90; d) Quantifying the polysorbate in the eluate of step c). In some embodiments, the polypeptide binds specifically and non-specifically to the chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95,96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, wherein the eluate from step c) comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) of the total polypeptides in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (e.g., less than about 4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% of any one or less) of total NAT in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of mobile phase B is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase a is about 10. In some embodiments, the second ratio is about 45. In some embodiments, the third ratio is about 100. In some embodiments, mobile phase a comprises an aqueous solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises an organic solvent (e.g., methanol or acetonitrile) solution of ammonium hydroxide in about 0.5% to about 5% (v/v) (e.g., any of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9, 1.1,1.3,1.4,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20, 25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8, 3,3.2,3.4,3.6,3.8,4,4.5, 5) minutes after step a) begins. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., any one or more of 2.2,2.4,2.6,2.8, 3,3.1,3.2,3.4,3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005,0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7, 0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., about any of 0.2, 0.5,1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the composition further comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., any of about 0.2, 0.5,1,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80, or 90mM, including any range between these values). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40, 60,80,100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4, 6.6,6.8,7.0,7.2,7.4, including any range between these values).

In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the polysorbate is fixedThe amount comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%, 0.5%,0.1%,0.05%,0.01% or less) interference from the polypeptide and NAT.

In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate, polypeptide, and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a of between about 5 to about 85, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises a methanol solution of ammonium hydroxide; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a of between about 40; c) Eluting the polysorbate from the chromatography material with a solution comprising mobile phase B to mobile phase a at a third ratio of between about 90; d) Quantifying the polysorbate in the eluate of step c), wherein the quantifying of the polysorbate comprises less than about 10% (e.g., less than about any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%, 0.5%,0.1%,0.05%,0.01% or less) interference from the polypeptide and NAT. In some embodiments, the polypeptide specifically and non-specifically binds to a chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95, 96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%, 0.5%,0.1%,0.05%,0.01% or less) of total polypeptides in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (e.g., less than about 4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% of any one or less) of total NAT in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is about 10. In some embodiments, the second ratio is about 45:55. In some embodiments, the third ratio is about 100. In some embodiments, mobile phase a comprises an aqueous solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises a methanolic solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.3,1.4,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8, 9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4, 1.5,1.6,1.7,1.8,1.9, 2) minutes after the beginning of step a) and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8,4,4.5, 5) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08,0.09,0.1,0.2,0.3, 0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., any one or more of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4,3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005,0.01,0.05,0.1,0.2, 0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the composition further comprises methionine. In some embodiments of the present invention, the substrate is, The concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8,9,10,20, 30,40,50,60,70,80, or 90mM, including any range therebetween). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4, 5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD).

In some embodiments, methods for quantifying the amount of a composition comprising a polysorbate, a polypeptide, and N-acetyltryptophan are providedThe method of polysorbate in a composition of (a), wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a of between about 5 to about 15; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a of between about 40; c) Eluting the polysorbate from the chromatographic material with a solution comprising mobile phase B to mobile phase a in a third ratio of between about 90; d) Quantifying polysorbate in the eluate of step c), wherein the eluate from step c) comprises less than about 10% (e.g., less than about any one or less of 9%,8%, 7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%, 0.01%) of total polypeptides in the composition and less than about 5% (e.g., less than about any one or less of 4%,3%,2%,1%,0.5%,0.1%,0.05%, 0.01%) of total NATs in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is about 10. In some embodiments, the second ratio is about 45. In some embodiments, the third ratio is about 100. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) (e.g., any one of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) ammonium hydroxide. In some embodiments, mobile phase B comprises a methanolic solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.3,1.4, 1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40, and 45, including any range between these values) ) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9, 1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.2, 3.4,3.6,3.8,4,4.5, 5) minutes after step a) begins. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08, 0.09,0.1,0.2,0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4, 3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005, 0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6, 7,8, or 9mM, including any range between these values). In some embodiments, the composition further comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6, 7,8,9,10,20,30,40,50,60,70,80, or 90mM, including any range therebetween). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120, 140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0, 7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic is The protein is polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, thiomab ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the polysorbate comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%, 0.01% or less) interference from the polypeptide.

In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate, polypeptide, and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a between about 5 to about 15, 85, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises an acetonitrile solution of ammonium hydroxide; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a of between about 40; c) Eluting from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase a of between about 90A polysorbate; d) Quantifying the polysorbate in the eluate of step c), wherein the quantifying of the polysorbate comprises less than about 10% (e.g., less than about any one of about 9%,8%,7%, 6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) interference from the polypeptide and NAT. In some embodiments, the polypeptide specifically and non-specifically binds to a chromatography material, and at least about 90% (e.g., at least any one of about 91,92,93,94,95, 96,97,98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises non-specifically bound polypeptides. In some embodiments, wherein the eluate from step c) comprises less than about 10% (e.g., less than any one of about 9%,8%,7%, 6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) of the total polypeptides in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (e.g., less than about 4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% of any one or less) of total NAT in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is about 10. In some embodiments, the second ratio is about 45. In some embodiments, the third ratio is about 100. In some embodiments, mobile phase a comprises an aqueous solution of ammonium hydroxide from about 0.5% to about 5% (v/v) (e.g., about any one of 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises from about 0.5% to about 5% (v/v) (e.g., any of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) ammonium hydroxide in acetonitrile. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.3,1.4,1.5,1.7,1.9, 2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2, 3,4,5,6,7,8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) is at least about after the beginning of step a) 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3, 1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes and for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8,4, 4.5, 5) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08,0.09,0.1,0.2, 0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., any one or more of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4,3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005,0.01,0.05,0.1,0.2, 0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the composition further comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8,9,10,20, 30,40,50,60,70,80, or 90mM, including any range between these values). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120,140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., any of about 4.6,4.8,5.0,5.2,5.4, 5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, monoclonal antibodies, humanized antibodies, human antibodies, chimeric antibodies Synthetic antibodies, multispecific antibodies, glycoengineered antibodies, antibody fragments, antibody drug conjugates, thiomab ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD).

In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate, polypeptide, and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising a first ratio of mobile phase B to mobile phase a between about 5 to about 15, 85, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises an acetonitrile solution of ammonium hydroxide; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase a of between about 40; c) Eluting the polysorbate from the chromatography material with a solution comprising mobile phase B to mobile phase a at a third ratio of between about 90; d) Quantifying polysorbate in the eluate of step c), wherein the eluate from step c) comprises less than about 10% (e.g., less than about any one of 9%,8%, 7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) of total polypeptides in the composition and less than about 5% (e.g., less) of total polypeptides in the composition About any one of 4%,3%,2%,1%,0.5%, 0.1%,0.05%,0.01% or less) of total NAT in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase a is about 10. In some embodiments, the second ratio is about 45. In some embodiments, the third ratio is about 100. In some embodiments, mobile phase a comprises an aqueous solution of about 0.5% to about 5% (v/v) (e.g., any one of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) ammonium hydroxide. In some embodiments, mobile phase B comprises from about 0.5% to about 5% (v/v) (e.g., any of about 0.75%,1%,1.5%,2%,2.5%,3%,3.5%,4%, and 4.5%, including any range between these values) ammonium hydroxide in acetonitrile. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.3,1.4, 1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., about any of 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9, 1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes after the beginning of step a) and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.2, 3.4,3.6,3.8,4,4.5, 5) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08, 0.09,0.1,0.2,0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4, 3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005, 0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, N- The concentration of acetyltryptophan ranges from about 0.1mM to about 10mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6, 7,8, or 9mM, including any range between these values). In some embodiments, the composition further comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6, 7,8,9,10,20,30,40,50,60,70,80, or 90mM, including any range between these values). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120, 140,160,180,200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0, 7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodimentsThe nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the polysorbate comprises less than about 10% (e.g., less than about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%, 0.01% or less) interference from the polypeptide.

In some embodiments, a method is provided for quantifying polysorbate 20 in a composition comprising polysorbate 20, a polypeptide, and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising mobile phase B and mobile phase a in a ratio of about 10; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising mobile phase B and mobile phase a in a ratio of about 45; c) Eluting polysorbate 20 from the chromatography material with a solution comprising mobile phase B to mobile phase a in a ratio of about 100; d) Quantifying polysorbate 20 in the eluate of step c). In some embodiments, the eluate from step c) comprises less than about 10% (e.g., less than any one of about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%, 0.01% or less) of total polypeptides in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (e.g., less than about 4%,3%,2%,1%,0.5%,0.1%,0.05%, 0.01% of any one or less) of total NAT in the composition. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.3, 1.4,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the flow rate for chromatography is about 1.40mL/min. In some embodiments, the volume of the composition applied to the chromatography material is from about 1 to about 50 (e.g., any of about 1, 2,3,4,5,6,7,8,9,10,15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, the volume of the composition applied to the chromatographic material is about 25 μ L. In some embodiments, the step Step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3, 1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes after the beginning of step a) and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8,4, 4.5, 5) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08,0.09,0.1,0.2, 0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., any one or more of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4,3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the concentration of polysorbate 20 in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005,0.01,0.05, 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the composition further comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8,9,10, 20,30,40,50,60,70,80, or 90mM, including any range between these values). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., any of about 2,5,10,20,40,60,80,100,120,140,160,180, 200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0, 5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody Antibodies, antibody fragments, antibody drug conjugates, thiomab ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of the polysorbate comprises less than about 10% (e.g., less than about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) interference from the polypeptide and NAT.

In some embodiments, a method is provided for quantifying polysorbate 20 in a composition comprising polysorbate 20, a polypeptide, and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising mobile phase B and mobile phase a in a ratio of about 10; b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising mobile phase B and mobile phase a in a ratio of about 45; c) Eluting polysorbate 20 from the chromatography material with a solution comprising mobile phase B to mobile phase a in a ratio of about 100; d) Quantifying polysorbate 20 in the eluate of step c). In some embodiments, the eluent from step c) comprises less than about 10% (e.g., less than about 9%,8%,7%,6%,5%,4%,3%,2%,1%, 0.5%,0.1%,0.05%, 0.01%) or less) of the total polypeptides in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (e.g., less than about 4%,3%,2%, 1%,0.5%,0.1%,0.05%,0.01% of any one or less) of total NAT in the composition. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., about any of 0.7,0.9,1.1,1.3, 1.4,1.5,1.7,1.9,2.1, and 2.3, including any range between these values) mL/min. In some embodiments, the flow rate for chromatography is about 1.40mL/min. In some embodiments, the volume of the composition applied to the chromatographic material is from about 1 to about 50 (e.g., any of about 1,2,3,4,5,6,7,8,9,10, 15,20,25,30,35,40, and 45, including any range between these values) μ L. In some embodiments, the volume of the composition applied to the chromatographic material is about 25 μ L. In some embodiments, step b) begins at least about 0.5 (e.g., at least about any one or more of 0.6,0.7,0.8,0.9,1,1.1,1.2,1.3, 1.4,1.5,1.6,1.7,1.8,1.9, 2) minutes after the beginning of step a) and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.2,3.4,3.6,3.8,4, 4.5, 5) minutes. In some embodiments, step c) begins at least about 0.05 (e.g., at least about any one or more of 0.06,0.07,0.08,0.09,0.1,0.2, 0.3,0.4,0.5,1,1.5, 2) minutes and continues for at least about 2 (e.g., at least about any one or more of 2.2,2.4,2.6,2.8,3,3.1,3.2,3.4,3.6,3.8,4,4.5, 5) minutes after the end of step b). In some embodiments, the concentration of polysorbate 20 in the composition is in the range of about 0.001% to 1.0% (w/v) (e.g., about any one of 0.005,0.01,0.05, 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM (e.g., about any of 0.2,0.5,1,2,3,4,5,6,7,8, or 9mM, including any range between these values). In some embodiments, the composition further comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM (e.g., any of about 0.2,0.5,1,2,3,4,5,6,7,8,9,10, 20,30,40,50,60,70,80, or 90mM, including these values Any range in between). In some embodiments, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL (e.g., about any of 2,5,10,20,40,60,80,100,120,140,160,180, 200,220, and 240mg/mL, including any range between these values). In some embodiments, the pH of the composition is from about 4.5 to about 7.5 (e.g., about any of 4.6,4.8,5.0, 5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM Or Thiomab ^TM A drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is

MCX chromatography material. In some embodiments, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD). In some embodiments, the quantification of polysorbate comprises less than about 10% (e.g., less than about 9%,8%,7%,6%,5%,4%,3%,2%,1%,0.5%,0.1%,0.05%,0.01% or less) interference from the polypeptide and NAT.

In some embodiments of any of the methods above, the non-ionic surfactant is quantified in a composition comprising the non-ionic surfactant prior to adding the polypeptide to the composition. In some embodiments, the concentration of the nonionic surfactant in the composition will be higher prior to addition of the polypeptide (e.g., the nonionic surfactant in the composition is diluted as the polypeptide is added). In some embodiments of any of the methods above, the non-ionic surfactant is quantified in a composition that comprises the non-ionic surfactant but no polypeptide. Such quantification may be used as a control or comparator for compositions comprising the nonionic surfactant and the polypeptide.

In some embodiments of any of the methods described above, a sample of the composition to be analyzed is added to an autosampler of a chromatography apparatus (e.g., an HPLC apparatus). In some embodiments, the sample in the autosampler is refrigerated (e.g., 5 ± 3 ℃). In some embodiments, one or more columns comprising a chromatography material are placed in a column chamber of a chromatography apparatus. In some embodiments, temperature control features can be employed to maintain the column chamber temperature within a narrow range (e.g., ± 1 ℃) from a set point during analysis. In some embodiments, the column effluent is monitored at 280 nm.

In some embodiments of any of the methods described above, a sample of the composition to be analyzed is diluted with the loading buffer to a concentration of the target polypeptide of about 0.1mg/mL to about 75mg/mL (e.g., about any of 0.2,0.4,0.6, 0.8,1,1.5,2,2.5,3,3.5,4,4.5,5,6,7,8,9,10,12,14,16, 18,20,25,30,35,40,45,50,55,60,65, or 70mg/mL, including any range between these values).

In some embodiments of any of the methods above, the chromatography instrument comprises a gradient pump (e.g., a low pressure quaternary gradient pump), an autosampler (e.g., an autosampler with temperature control capability), a column chamber (e.g., a thermally controlled column chamber), a UV detector (e.g., a diode array UV detector), and an Evaporative Light Scattering Detector (ELSD). In some embodiments, the chromatography instrument further comprises a pH and conductivity monitor (e.g., PCM-3000) to collect pH and conductivity data in real time. Instrument control, data acquisition and data analysis are performed using appropriate software (e.g., JMP 10).

In some embodiments of the invention, the ionic strength of the mobile phase, e.g., the elution buffer, is measured by the conductivity of the mobile phase. Conductivity refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In solution, current flows by ionic transport. Thus, as the amount of ions present in the aqueous solution increases, the solution will have a higher conductivity. The basic units of measurement for conductivity are siemens (or mho), mho (mS/cm), and can be measured using conductivity meters, such as various models of Orion conductivity meters. Since electrolytic conductivity is the ability of ions in a solution to carry current, the conductivity of a solution can be altered by changing the concentration of ions therein. For example, the concentration of the buffer and/or the concentration of the salt (e.g., sodium chloride, sodium acetate, or potassium chloride) in the solution can be varied to obtain the desired conductivity. Preferably, the salt concentration of each buffer is varied to achieve the desired conductivity.

In some embodiments, the mobile phase of the chromatography has an initial conductivity of greater than about any of 0.0mS/cm,0.5mS/cm, 1.0mS/cm,1.5mS/cm,2.0mS/cm,2.5mS/cm,3.0mS/cm,3.5mS/cm, 4.0mS/cm,4.5mS/cm,5.0mS/cm,5.5mS/cm,6.0mS/cm,6.5mS/cm, 7.0mS/cm,7.5mS/cm,8.0mS/cm,8.5mS/cm,9.0mS/cm,9.5mS/cm, 10mS/cm,11mS/cm,12mS/cm,13mS/cm,14mS/cm,15mS/cm,16mS/cm, 17.0mS/cm,18.0mS/cm,19 mS/cm, 0mS/cm, or 20 mS/cm. In some embodiments, the conductivity of the mobile phase is increased during chromatography, for example, by an ionic strength gradient. In some embodiments, the conductivity of the mobile phase at the completion of elution is greater than any one of about 1.0mS/cm,1.5mS/cm,2.0mS/cm,2.5mS/cm,3.0mS/cm,3.5mS/cm, 4.0mS/cm,4.5mS/cm,5.0mS/cm,5.5mS/cm,6.0mS/cm,6.5mS/cm, 7.0mS/cm,7.5mS/cm,8.0mS/cm,8.5mS/cm,9.0mS/cm,9.5mS/cm, 10mS/cm,11mS/cm,12mS/cm,13mS/cm,14mS/cm,15mS/cm,16mS/cm, 17.0mS/cm,18.0mS/cm,19.0mS/cm, or 20 mS/cm. In some embodiments, the conductivity of the mobile phase is increased by a linear gradient. In some embodiments, the conductivity of the mobile phase is increased by a step gradient comprising one or more steps.

In some embodiments of any of the methods described herein, the composition comprising the polypeptide and the nonionic surfactant is loaded onto the chromatographic material in an amount of greater than about 1,2,3,4,5,6,7,8,9,10,15,20,25,50, 100,200,300,400,500,600,700,800,900,1000,2000,3000,4000, 5000,6000,7000,8000,9000, or 10000 μ g of the polypeptide. In some embodiments, the composition is applied to the chromatographic material at a concentration of greater than about any of 0.5,1,1.5,2, 2.5,5,10,20,30,40,50,60,70,80,90,100,110,120,130,140, or 150 mg/mL. In some embodiments, the composition is diluted prior to loading onto the chromatographic material; for example, the ratio of 1,1. In some embodiments, the composition is diluted into the mobile phase of chromatography. In some embodiments, the composition is diluted into a loading buffer.

In some embodiments of the methods described herein, the chromatography material is in a column or cartridge. In some embodiments, the column is an HPLC column or cartridge. The column or cartridge may be of any size compatible with chromatography instrumentation. For example, in some embodiments, the column or cylinder has any one of the dimensions 2.1X 2 mm, 4X 50mm, 4X 100mm, 4X 150mm, 4X 200mm, 4X 250mm, or 2X 250mm.

III. Polypeptides

The polypeptides are provided for use in any ion exchange chromatography method, wherein the separation conditions are optimized as described herein. In some embodiments of the invention, the composition of the polypeptide is analyzed by ion exchange chromatography. Such methods can be used to identify charge variants of polypeptides in a composition. In some embodiments, the polypeptide is an antibody or fragment thereof. In some embodiments, the pI of the polypeptide is about 6.0 to about 9.5. In some embodiments, the polypeptide is an antibody having a pI ranging from about 6.0 to about 9.5. In some embodiments, the Inflection Point (IP) of the charge versus pH curve of a polypeptide is provided by the methods of the invention. In some embodiments, the change in IP with temperature (dIP/dT) is provided by the methods of the invention.

In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the polypeptide is an antibody. In some embodiments, the polypeptide is an immunoadhesin.

In some embodiments, the polypeptide has a molecular weight greater than any of about 5,000 daltons, 10,000 daltons, 15,000 daltons, 25,000 daltons, 50,000 daltons, 75,000 daltons, 100,000 daltons, 125,000 daltons, or 150,000 daltons. The polypeptide can have a molecular weight of between about 50,000 daltons and 200,000 daltons or between 100,000 daltons and 200,000 daltons. Alternatively, the polypeptides used herein may have a molecular weight of about 120,000 daltons or about 25,000 daltons.

pI is the isoelectric point and is the pH at which a particular molecule or surface carries no net charge. In some embodiments, the methods of the invention are useful for a variety of compositions comprising polypeptides, wherein the pI of the polypeptides, e.g., antibodies, in the composition ranges from about 6.0 to about 9.5. In some embodiments, the pI of the polypeptide is greater than about 9.5; for example, from about 9.5 to about 12. In some embodiments of any of the methods described herein, the pI of the polypeptide, e.g., antibody, can be less than about 7; for example, from about 4 to about 7.

In an embodiment of any of the methods described herein, the one or more contaminants in the composition comprising the polypeptide and the one or more contaminants are polypeptide charge variants. In some embodiments, a polypeptide charge variant is a polypeptide that has been modified from its native state, thereby altering the charge of the polypeptide. In some embodiments, the charge variant is more acidic than the parent polypeptide; i.e., having a lower pI than the parent polypeptide. In other embodiments, the charge variant is more basic than the parent polypeptide; i.e., having a higher pI than the parent polypeptide. In some embodiments, the polypeptide charge variant is engineered. In some embodiments, the polypeptide charge variant is the result of a natural process; for example, oxidation, deamidation, C-terminal processing of lysine residues, N-terminal pyroglutamate formation and saccharification. In some embodiments, the polypeptide charge variant is a glycoprotein, wherein a glycan attached to the protein is modified such that the charge of the glycoprotein is altered as compared to the parent glycoprotein; for example by addition of sialic acid or derivatives thereof. In some embodiments, the polypeptide charge variant is an antibody charge variant.

The polypeptides analyzed using the methods described herein are typically produced using recombinant techniques. Methods of producing recombinant proteins are described, for example, in U.S. Pat. nos. 5,534,615 and 4,816,567, which are expressly incorporated herein by reference. In some embodiments, the protein of interest is produced in CHO cells (see, e.g., WO 94/11026). In some embodiments, the polypeptide of interest is produced in an escherichia coli cell. See, for example, U.S. Pat. nos. 5,648,237; U.S. Pat. No. 5,789,199 and U.S. Pat. No. 5,840,523, which describe Translation Initiation Regions (TIR) and signal sequences for optimized expression and secretion. See also Charlton, methods in Molecular Biology, vol.248 (b.k. C.lo, ed., humana Press, totowa, n.j., 2003), pp.245-254, which describes the expression of antibody fragments in e.coli. When recombinant techniques are used, the polypeptide may be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium.

The polypeptide may be recovered from the culture medium or the host cell lysate. Cells used to express the polypeptide may be disrupted by a variety of physical or chemical methods, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents. If the polypeptide is produced intracellularly, as a first step, particulate debris of the host cells or of the lysed fragments is removed, for example by centrifugation or ultrafiltration. Carter et al, bio/Technology 10: 163-167 (1992) describes a method for isolating polypeptides secreted into the periplasmic space of E.coli. Briefly, the cell paste was thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) in about 30 minutes. Cell debris can be removed by centrifugation. In the case of secretion of the polypeptide into the culture medium, the supernatant from such expression systems is usually first concentrated using a commercially available polypeptide concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

In some embodiments, the polypeptide in the composition comprising the polypeptide and one or more contaminants has been purified or partially purified prior to analysis by the methods of the invention. For example, the polypeptide of the method is in an eluate from affinity chromatography, cation exchange chromatography, anion exchange chromatography, mixed mode chromatography and hydrophobic interaction chromatography. In some embodiments, the polypeptide is in an eluate from protein a chromatography.

Examples of polypeptides that can be analyzed by the methods of the invention include, but are not limited to, immunoglobulins, immunoadhesins, antibodies, enzymes, hormones, fusion proteins, fc-containing proteins, immunoconjugates, cytokines and interleukins.

(A) Antibodies

In some embodiments of any of the methods described herein, the polypeptide of any of the methods for analyzing polypeptides and preparations comprising polypeptides by the methods described herein is an antibody.

Molecular targets of antibodies include (i) CD proteins and their ligands, such as, but not limited to, CD3, CD4, CD8, CD19, CD11a, CD20, CD22, CD27, CD28, CD34, CD40, CD79a (CD 79 a), CD79 β (CD 79 b), CD122, and CD137; (ii) Cytokines such as, but not limited to, IL-13, IL-17, IL-22, and IL-33; (iii) A member of the ErbB receptor family, such as the EGF receptor, the HER2, HER3 or HER4 receptor; (iv) Cell adhesion molecules such as LFA-1, mac1, p150,95, VLA-4, ICAM-1, VCAM and α v/β 3 integrins, including their α or β subunits (e.g., anti-CD 11a, anti-CD 18 or anti-CD 11b antibodies); (v) growth factors such as VEGF; TGF β, igE; blood group antigens; the flk2/flt3 receptor; obesity (OB) receptors; an mpl receptor; CTLA-4; protein C, BR3, C-met, tissue factor,. Beta.7, etc.; (vi) Immunomodulatory proteins, such as OX40, GITR, ICOS, PD-1, PD-L2, LAG3, TIM-3, and VISTA; (vii) Cell surface and transmembrane Tumor Associated Antigens (TAAs), such as those described in U.S. patent No. 7,521,541, include, but are not limited to, naPi2b.

Other exemplary antibodies include those selected from, but not limited to: anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-P53 antibody, anti-HER-2/neu antibody, anti-EGFR antibody, anti-TGF β antibody, anti-OX 40 antibody, anti-GITR antibody, anti-ICOS antibody, anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L2 antibody, anti-TIM-3 antibody, anti-VISTA antibody, anti-cathepsin D antibody, anti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA 125 antibody, anti-CA 15-3 antibody, anti-CA 19-9 antibody, anti-c-B-2 antibody, anti-P-glycoprotein antibody, anti-CEA antibody, anti-retinoblastoma protein antibody, anti-RAs oncoprotein antibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD 3 antibody, anti-CD 4 antibody, anti-CD 5 antibody, anti-CD 7 antibody, anti-CD 8 antibody, anti-CD 9/P24 antibody, anti-CD 10 antibody, anti-CD 11a antibody, anti-CD 11c antibody, anti-CD 13 antibody, anti-CD 14 antibody, anti-CD 15 antibody, anti-CD 19 antibody, anti-CD 20 antibody, anti-CD 22 antibody, anti-CD 23 antibody, anti-CD 27 antibody, anti-CD 28 antibody, anti-CD 30 antibody, anti-CD 31 antibody, anti-CD 33 antibody, anti-CD 34 antibody, anti-CD 35 antibody, anti-CD 38 antibody, anti-CD 40 antibody, anti-CD 41 antibody, anti-LCA/CD 45 antibody, anti-CD 45RO antibody, anti-CD 45RA antibody, anti-CD 39 antibody, anti-CD 100 antibody, anti-CD 95/Fas antibody, anti-CD 99 antibody, anti-CD 106 antibody, anti-CD 122 antibody, anti-CD 137 antibody, anti-ubiquitin antibody, anti-CD 71 antibody, anti-StafcrA antibody, anti-H5 antibody, anti-Ly 6E antibody, anti-STEAP antibody, anti-FluB antibody, anti-VEGF antibody, anti-Ang 2 antibody, anti-FGFR 1 antibody, anti-KLB antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-vimentin antibody, anti-HPV protein antibody, anti-kappa light chain antibody, anti-lambda light chain antibody, anti-melanosome antibody, anti-prostate specific antigen antibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody, anti-Tn antigen antibody, and MetMab.

(i) Monoclonal antibodies

In some embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, i.e., each antibody comprised in the population is identical and/or binds the same epitope, these variants usually being present in minor amounts, except for possible variants produced during the production of the monoclonal antibody. Thus, the modifier "monoclonal" indicates that the antibody is not characterized as a mixture of discrete or polyclonal antibodies.

For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al, nature 256 (1975), or can be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other suitable host animal, e.g., a hamster, is immunized as described herein to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the polypeptide for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, monoclonal Antibodies: principles and Practice, pp.59-103 (Academic Press, 1986)).

The hybridoma cells so prepared are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

In some embodiments, the myeloma cells are efficiently fused cells, support stable high levels of antibody production by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, in some embodiments, the myeloma Cell line is a murine myeloma Cell line, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, san Diego, california USA, and SP-2 or X63-Ag8-653 cells available from American Type Culture Collection, rockville, maryland USA. Human myeloma and mouse-human heteromyeloma cell lines have been described for the Production of human Monoclonal antibodies (Kozbor, J.Immunol. 133 (1984); brodeur et al, monoclonal Antibody Production Techniques and Applications pp.51-63 (Marcel Dekker, inc., new York, 1987)).

Determining the production of monoclonal antibodies directed against the antigen in the medium in which the hybridoma cells are grown. In some embodiments, the binding specificity of a monoclonal antibody produced by a hybridoma cell is determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis of Munson et al, anal. Biochem.107:220 (1980).

After identification of hybridoma cells producing Antibodies with the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and cultured by standard methods (Goding, monoclonal Antibodies: principles and Practice pp.59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can grow in vivo as ascites tumors in the animal.

Monoclonal antibodies secreted by subclones were purified by conventional immunoglobulin purification procedures (e.g., polypeptide A-

Hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography) is suitably separated from the culture medium, ascites fluid or serum.

DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). In some embodiments, hybridoma cells are used as a source of such DNA. Once isolated, the DNA may be placed into an expression vector and then transfected into a host cell, such as an E.coli cell, simian COS cell, chinese Hamster Ovary (CHO) cell, or myeloma cell that does not produce immunoglobulin polypeptides, to obtain the synthesis of monoclonal antibodies in the recombinant host cell. Review articles on recombinant expression of DNA encoding antibodies in bacteria include Skerra et al, curr. Opinion in immune. 5 (1993) and pluckthun, immune. Revs.,130 (1992).

In another embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using techniques described in McCafferty et al, nature 348. Clackson et al, nature 352-628 (1991) and Marks et al, J. Mol. Biol.222:581-597 (1991) describe the use of phage libraries to isolate murine and human antibodies, respectively. Subsequent publications describe the generation of high affinity (nM range) human antibodies by chain shuffling (Marks et al, bio/Technology 10. Thus, these techniques are viable alternatives to conventional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

The DNA may also be modified, for example, by replacing homologous murine sequences with the coding sequences for human heavy and light chain constant domains (U.S. Pat. nos. 4,816,567, morrison et al, proc.natl acad.sci.usa 81 (1984)), or by covalently linking the immunoglobulin coding sequence to all or part of the coding sequence for a non-immunoglobulin polypeptide.

Typically, such non-immunoglobulin polypeptides replace the constant domains of an antibody, or they replace the variable domains of one antigen binding site of an antibody to produce a chimeric bivalent antibody comprising one antigen binding site specific for one antigen and another antigen binding site specific for a different antigen.

In some embodiments of any of the methods described herein, the antibody is IgA, igD, igE, igG, or IgM. In some embodiments, the antibody is an IgG monoclonal antibody.

(ii) Humanized antibodies

In some embodiments, the antibody is a humanized antibody. Methods for humanizing non-human antibodies have been described in the art. In some embodiments, the humanized antibody has one or more amino acid residues from a non-human source introduced into it. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be performed essentially according to the method of Winter and co-workers (Jones et al, nature 321-522-525 (1986); riechmann et al, nature 332 323-327 (1988); verhoeyen et al, science 239 1534-1536 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains (light and heavy chains) for making humanized antibodies is important for reducing antigenicity. According to the so-called "best fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence closest to the rodent was then accepted as the human Framework Region (FR) of the humanized antibody (Sims et al, J. Immunol.151:2296 (1993); chothia et al, J. Mol. Biol. 196 (1987)). Another approach uses specific framework regions derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chain variable regions. The same framework can be used for several different humanized antibodies (Carter et al, proc. Natl. Acad. Sci. USA 89 (1992); presta et al, J. Immunol.151:2623 (1993)).

More importantly, the antibodies are humanized, retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, in some embodiments of the method, humanized antibodies are made by methods of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. A computer program is available to elucidate and display the possible three-dimensional conformational structures of the selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected and combined from the recipient and import sequences to achieve a desired antibody characteristic, such as increased affinity for the target antigen. Typically, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

(iii) Human antibodies

In some embodiments, the antibody is a human antibody. As an alternative to humanization, human antibodies can be produced. For example, it is now possible to produce transgenic animals (e.g., mice) that, upon immunization, are capable of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, antibody heavy chain joining regions (J) have been described in chimeric and germline mutant mice _H ) Homozygous deletion of the gene results in complete inhibition of endogenous antibody production. Transfer of human species in such germ line mutant miceImmunoglobulin gene arrays will result in the production of human antibodies following antigen challenge. See, e.g., jakobovits et al, proc.natl.acad.sci.usa 90 (1993); jakobovits et al, nature362:255-258 (1993); bruggermann et al, yeast in Immuno. 7 (1993); and U.S. Pat. nos. 5,591,669;5,589,369; and 5,545,807.

Alternatively, phage display technology (McCafferty et al, nature 348 (1990)) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene libraries from uninmmunized donors. According to this technique, antibody V domain genes are cloned in-frame into the major or minor coat polypeptide genes of filamentous phage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody displaying these properties. Thus, the phage mimics some of the properties of B cells. Phage display can be performed in a variety of formats; for a review, see, e.g., johnson, kevin S.and Chiswell, david J., current Opinion in Structural Biology 3. Several sources of V gene segments are available for phage display. Clackson et al, nature 352, 624-628 (1991) isolated multiple antibodies from a small random combinatorial library of V genes derived from the spleen of immunized mice

An oxazolone antibody. A V gene bank from an unimmunized human donor can be constructed and antibodies to a variety of antigens, including self-antigens, can be isolated essentially as described in Marks et al, J.mol.biol. 222. See also U.S. Pat. nos. 5,565,332 and 5,573,905.

Human antibodies can also be produced by activated B cells in vitro (see U.S. Pat. nos. 5,567,610 and 5,229,275).

(iv) Antibody fragments

In some embodiments, the antibody is an antibody fragment. Various techniques have been developed for the production of antibody fragments. Conventionally, these areFragments are obtained by proteolytic digestion of the intact antibody (see, e.g., morimoto et al, journal of Biochemical and Biophysical Methods 24, 107-117 (1992) and Brennan et al, science 229 (1985). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, fab '-SH fragments can be recovered directly from E.coli and chemically coupled to form F (ab') ₂ Fragment (Carter et al, bio/Technology 10 (1992). According to another method, F (ab') can be isolated directly from recombinant host cell cultures ₂ And (3) fragment. Other techniques for producing antibody fragments will be apparent to the skilled person. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. nos. 5,571,894; and U.S. Pat. No. 5,587,458. The antibody fragment may also be a "linear antibody," e.g., as described in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

In some embodiments, fragments of the antibodies described herein are provided. In some embodiments, the antibody fragment is an antigen-binding fragment. In some embodiments, the antigen binding fragment is selected from the group consisting of a Fab fragment, a Fab 'fragment, F (ab') ₂ Fragments, scFv, fv and diabodies.

(v) Bispecific antibodies

In some embodiments, the antibody is a bispecific antibody. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. An exemplary bispecific antibody can bind two different epitopes. Alternatively, the bispecific antibody binding arms can be combined with arms that bind to trigger molecules on leukocytes, such as T cell receptor molecules (e.g., CD2 or CD 3), or Fc receptors of IgG (Fc γ R), such as Fc γ RI (CD 64), fc γ RII (CD 32), and Fc γ RIII (CD 16), in order to focus cellular defense mechanisms on the cell. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F (ab') ₂ Bispecific antibodies).

Methods of making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al, nature 305. Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, usually by an affinity chromatography step, is rather cumbersome and the product yield is low. Similar methods are disclosed in WO 93/08829 and Trunecker et al, EMBO J., 10.

According to different methods, antibody variable domains (antibody-antigen binding sites) with the desired binding specificity are fused to immunoglobulin constant domain sequences. In some embodiments, the fusion is with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2, and CH3 regions. In some embodiments, a first heavy chain constant region (CH 1) containing the site necessary for light chain binding is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in an embodiment, as the unequal ratios of the three polypeptide chains used in the construction provide the best yield. However, when expressing at least two polypeptide chains in equal ratios results in high yields or when the ratio is not particularly important, the coding sequences for two or all three polypeptide chains can be inserted into one expression vector.

In some embodiments of this method, the bispecific antibody consists of a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure was found to facilitate the separation of the desired bispecific compound in combination with the undesired immunoglobulin chain, since the presence of the immunoglobulin light chain in only half of the bispecific molecule provides an easy way of separation. This method is disclosed in WO 94/04690. For further details on the generation of bispecific antibodies, see, e.g., suresh et al, methods in Enzymology 121 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. In some embodiments, the interface comprises a C of an antibody constant domain _H 3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By substituting a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size to the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers relative to other undesired end products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody in the heterologous conjugate can be coupled to avidin and the other to biotin. For example, such antibodies have been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and are useful in the treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). The heteroconjugate antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents, as well as a number of crosslinking techniques, are well known in the art and are disclosed in U.S. Pat. No. 4,676,980.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, science 229: 81 (1985) describes a method in which intact antibodies are proteolytically cleaved to yield F (ab') ₂ And (3) fragment. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab ' -TNB derivatives is then reconverted to the Fab ' -thiol by reduction with mercaptoethylamine and with equimolar amounts of the other Fab ' -TN The B derivatives are mixed to form the bispecific antibody. The bispecific antibodies produced can be used as reagents for the selective immobilization of enzymes.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell cultures are also described. For example, bispecific antibodies are produced using leucine zippers. Kostelny et al, J. Immunol.148 (5): 1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. The method can also be used to produce antibody homodimers. The "diabody" technique described by Hollinger et al, proc.natl.acad.sci.usa 90. The fragments comprise a light chain variable domain (V) joined by a linker _L ) Linked heavy chain variable domains (V) _H ) The linker is too short to allow pairing between two domains on the same strand. Thus, V of a segment is forced _H And V _L Complementarity of the Domain to the other fragment V _L And V _H The domains pair, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by using single chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol.152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al, J.Immunol.147:60 (1991).

(vi) Multivalent antibodies

In some embodiments, the antibody is a multivalent antibody. Multivalent antibodies can be internalized (and/or catabolized) faster than bivalent antibodies by cells expressing the antigen to which the antibodies bind. The antibodies provided herein can be multivalent antibodies (other than IgM classes) with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acids encoding the polypeptide chains of the antibody. A multivalent antibody may comprise a dimerization domain and three or more antigen binding sites. Preferred dimerization domains comprise (or consist of) an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. Preferred multivalent antibodies herein comprise (or consist of) three to about eight, but preferably four antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (preferably two polypeptide chains), wherein the polypeptide chain comprises two or more variable domains. For example, the polypeptide chain can comprise VD1- (X1) n-VD2- (X2) n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, and Fc is one polypeptide chain of an Fc region. X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain can comprise a VH-CH 1-flexible linker-VH-CH 1-Fc region chain; or a chain of VH-CH1-VH-CH1-Fc regions. The multivalent antibody herein preferably further comprises at least two (preferably four) light chain variable domain polypeptides. Multivalent antibodies herein may, for example, comprise about 2 to about 8 light chain variable domain polypeptides. Light chain variable domain polypeptides contemplated herein comprise a light chain variable domain, and optionally, further comprise a CL domain.

In some embodiments, the antibody is a multispecific antibody. Examples of multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (V) _H ) And a light chain variable domain (V) _L ) The antibody of (1), wherein V _H V _L The unit has multi-epitope specificity; having two or more V _L And V _H Antibodies of the Domain, each V _H V _L The units bind different epitopes; an antibody having two or more single variable domains, each single variable domain binding a different epitope; a full-length antibody; antibody fragments such as Fab, fv, dsFv, scFv; diabodies, bispecific diabodies, triabodies, trifunctional antibodies, covalently or non-covalently linked antibody fragments. In some embodiments, the antibody has polyepitopic specificity; for example, the ability to specifically bind two or more different epitopes on the same or different targets. In some embodiments, the antibody is monospecific; for example, an antibody that binds only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that binds each epitope with an affinity of 5 μ M to 0.001pm,3 μ M to 0.001pM, 1 μ M to 0.001pm,0.5 μ M to 0.001pM, or 0.1 μ M to 0.001 pM.

(vii) Other antibody modifications

It may be desirable to modify the antibodies provided herein with respect to effector function, e.g., to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibodies. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or additionally, cysteine residues may be introduced in the Fc region, allowing interchain disulfide bonds to form in this region. The homodimeric antibody thus produced may have improved internalization capacity and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, J.exp Med.176:1191-1195 (1992) and shop, B.J., immunol.148. Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al, cancer Research 53. Alternatively, antibodies with dual Fc regions can be engineered to have enhanced complement-mediated lysis and ADCC capabilities. See Stevenson et al, anti-Cancer Drug Design 3.

To increase the serum half-life of an antibody, amino acid changes may be made in the antibody, as described in US 2006/0067930, which is incorporated herein by reference in its entirety.

(B) Polypeptide variants and modifications

Amino acid sequence modifications of polypeptides (including antibodies) described herein can be used in methods of purifying polypeptides (e.g., antibodies) described herein.

(i) Variant polypeptides

By "polypeptide variant" is meant a polypeptide, preferably an active polypeptide, as defined herein, which has at least about 80% amino acid sequence identity to the full-length native sequence of the polypeptide; a polypeptide sequence lacking a signal peptide; the extracellular domain of the polypeptide with or without a signal peptide. Such polypeptide variants include, for example, polypeptides having one or more amino acid residues added or deleted at the N-or C-terminus of the full-length native amino acid sequence. Typically, a TAT polypeptide variant will have at least about 80% amino acid sequence identity to the full-length native sequence polypeptide sequence, the polypeptide sequence lacking a signal peptide, the extracellular domain of the polypeptide with or without a signal peptide, or at least about 85%,90%,95%,96%,97%,98%, or 99% amino acid sequence identity of either. Optionally, the variant polypeptide will have no more than one conservative amino acid substitution as compared to the native polypeptide sequence, or no more than any one of about 2,3,4,5,6,7,8,9, or 10 conservative amino acid substitutions as compared to the native polypeptide sequence.

Variant polypeptides may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared to the full-length native polypeptide. Certain variant polypeptides may lack amino acid residues that are not essential for the desired biological activity. These variant polypeptides having truncations, deletions, and insertions can be prepared by any of a number of conventional techniques. The desired variant polypeptide may be chemically synthesized. Another suitable technique involves isolating and amplifying nucleic acid fragments encoding the desired variant polypeptides by Polymerase Chain Reaction (PCR). Oligonucleotides that define the desired ends of the nucleic acid fragments are used for the 5 'and 3' primers in PCR. Preferably, the variant polypeptide shares at least one biological and/or immunological activity with a native polypeptide disclosed herein.

Amino acid sequence insertions include amino-and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion of the N-or C-terminus of the antibody with an enzyme or polypeptide that increases the serum half-life of the antibody.

For example, it may be desirable to improve the binding affinity and/or other biological properties of a polypeptide. Amino acid sequence variants of a polypeptide are prepared by introducing appropriate nucleotide changes into antibody nucleic acids or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequence of the polypeptide. If the final construct has the desired characteristics, any combination of deletions, insertions and substitutions are made to arrive at the final construct. Amino acid changes can also alter post-translational processes of a polypeptide (e.g., an antibody), such as changing the number or position of glycosylation sites.

By comparing the sequence of the polypeptide with the sequence of homologous known polypeptide molecules and minimizing the number of amino acid sequence changes made in regions of high homology, guidance can be found in determining which amino acid residues can be inserted, substituted or deleted without adversely affecting the desired activity.

A useful method for identifying certain residues or regions of a polypeptide (e.g., an antibody) as preferred positions for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells, science 244, 1081-1085 (1989). Here, a residue or set of target residues (e.g., charged residues such as Arg, asp, his, lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Those amino acid positions that demonstrate functional sensitivity to substitution are then improved by introducing further or other variants at or for the substitution site. Thus, although the site at which the amino acid sequence variation is introduced is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is performed at the target codon or region and the expressed antibody variants are screened for the desired activity.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule substituted with a different residue. Sites of most interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown below in table 1 under the heading of "conservative substitutions". If such substitutions result in a change in biological activity, more substantial changes, referred to in Table 1 as "exemplary substitutions" or as further described below with reference to amino acid classes, can be introduced and the products screened.

TABLE 1

Substantial modification of the biological properties of a polypeptide is achieved by selecting substitutions that differ significantly in the effect of maintaining (a) the structure of the polypeptide backbone in the region of the substitution (e.g., a sheet or helical conformation), (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Amino acids can be grouped according to similarity in their side chain properties (A.L. Lehninger, biochemistry second, pp.73-75, worth publications, new York (1975)):

(1) Apolar Ala (A), val (V), leu (L), ile (I), pro (P), phe (F), trp (W), met (M)

(2) Uncharged polarity Gly (G), ser (S), thr (T), cys (C), tyr (Y), asn (N), gln (Q)

(3) Acidic Asp (D), glu (E)

(4) Basic Lys (K), arg (R), his (H)

Alternatively, naturally occurring residues may be grouped into groups based on common side chain properties:

(1) Hydrophobicity norleucine, met, ala, val, leu, ile;

(2) Neutral hydrophilic Cys, ser, thr, asn, gln;

(3) Asp and Glu as acidity;

(4) Basic His, lys, arg;

(5) Residues affecting chain orientation Gly, pro;

(6) Aromatic, trp, tyr, phe.

Non-conservative substitutions will require the exchange of a member of one of these classes for a member of the other class.

Any cysteine residues not involved in maintaining the correct conformation of the antibody may also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds may be added to the polypeptide to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized antibody). Typically, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they were produced. A convenient way to generate such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) were mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues which significantly contribute to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the points of contact between the antibody and the target. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are produced, the set of variants is screened as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

Another amino acid variant of the polypeptide alters the original glycosylation pattern of the antibody. The polypeptide may comprise non-amino acid moieties. For example, the polypeptide may be glycosylated. Such glycosylation may occur naturally during expression of the polypeptide in the host cell or host organism, or may be a deliberate modification by human intervention. Alteration refers to deletion of one or more carbohydrate moieties found in the polypeptide, and/or addition of one or more glycosylation sites not present in the polypeptide.

Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly to serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to the polypeptide is conveniently accomplished by altering the amino acid sequence to contain one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Changes may also be made by the addition or substitution of one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

Removal of the carbohydrate moiety present on the polypeptide may be accomplished by chemical or enzymatic methods or by mutational substitution of codons encoding amino acid residues that are targets of glycosylation. Enzymatic cleavage of the carbohydrate moiety on the polypeptide can be achieved by using a variety of endoglycosidases and exoglycosidases.

Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine and histidine side chains, acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

(ii) Chimeric polypeptides

The polypeptides described herein may be modified in a manner to form a chimeric molecule comprising a polypeptide fused to another heterologous polypeptide or amino acid sequence. In some embodiments, the chimeric molecule comprises a fusion of a polypeptide and a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. Epitope tags are typically located at the amino or carboxy terminus of the polypeptide. The presence of such epitope-tagged forms of the polypeptide can be detected using antibodies directed against the tag polypeptide. Furthermore, the provision of an epitope tag enables the polypeptide to be easily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds the epitope tag.

In an alternative embodiment, the chimeric molecule may comprise a fusion of the polypeptide with an immunoglobulin or a specific region of an immunoglobulin. The bivalent form of the chimeric molecule is called "immunoadhesin".

As used herein, the term "immunoadhesin" refers to antibody-like molecules that combine the binding specificity of a heterologous polypeptide with the effector functions of an immunoglobulin constant domain. Structurally, immunoadhesins comprise fusions of amino acid sequences other than the antigen recognition and binding site of an antibody (i.e., "heterologous") and immunoglobulin constant domain sequences with the desired binding specificity. The adhesin part of an immunoadhesin molecule is typically a contiguous amino acid sequence comprising at least a binding site for a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin can be obtained from any immunoglobulin, for example, igG-1, igG-2, igG-3 or IgG-4 subtypes, igA (including IgA-1 and IgA-2), igE, igD or IgM.

Ig fusions preferably include substitutions in the soluble (transmembrane domain deleted or inactivated) form of the polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion comprises the hinge, CH, of an IgG1 molecule ₂ And CH ₃ Or a hinge, CH ₁ ，CH ₂ And CH ₃ And (4) a zone.

(iii) Polypeptide conjugates

The polypeptides used in the polypeptide formulations may be conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof) or a radioisotope (i.e., a radioconjugate).

Chemotherapeutic agents useful for producing such conjugates may be used. In addition, enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, anemonin II A chain, alpha-sarcin, aleurites fordii protein, dianthin protein, pokeweed (Phytolacca americana) protein (PAPI, PAPII and PAP-S), matrine inhibitor, curcin, crotin, saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and tricothecenes. A variety of radionuclides are useful for producing radioconjugated polypeptides. Examples include ²¹² Bi, ¹³¹ I,131In,90Y, and 186Re. Conjugates of a polypeptide and a cytotoxic agent are prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutathione), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-azido derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene). For example, a ricin immunotoxin may be prepared as described in Vitetta et al, science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating a radioactive nucleotide to a polypeptide.

Also contemplated herein are conjugates of polypeptides and one or more small molecule toxins (e.g., calicheamicin, maytansinoids, triterpenes and CC 1065) as well as toxin-active derivatives of these toxins.

Maytansinoids are mitotic inhibitors, which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata. Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters. Synthetic maytansinol and derivatives and analogues thereof are also contemplated. Many linking groups are known in the art for use in the preparation of polypeptide-maytansinoid conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020. The linking group includes a disulfide bond group, a thioether group, an acid labile group, a photolabile group, a peptidase labile group or an esterase labile group, as disclosed in the above patents, and preferably a disulfide bond and a thioether group.

Depending on the type of linkage, the linker can be attached to the maytansinoid molecule at multiple positions. For example, ester linkages can be formed by reaction with hydroxyl groups using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with a hydroxymethyl group, the C-15 position modified with a hydroxyl group and the C-20 position having a hydroxyl group. In a preferred embodiment, the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue.

Another conjugate of interest comprises a polypeptide conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see, e.g., U.S. Pat. No. 5,712,374. Structural analogs of calicheamicin that may be used include, but are not limited to, gamma ₁ I,α ₂ I,α ₃ ^I N-acetyl-gamma ₁ ^I PSAG and θ ₁ ^I . Another antineoplastic drug to which antibodies may be conjugated is QFA, which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Thus, cellular uptake of these agents by polypeptide (e.g., antibody) mediated internalization greatly enhances their cytotoxic effects.

Other anti-tumor agents that can be conjugated to the polypeptides described herein include BCNU, streptozotocin, vincristine and 5-fluorouracil, a family of agents known collectively as the LL-E33288 complex, and esperamicins.

In some embodiments, the polypeptide can be a conjugate between the polypeptide and a compound having nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease, e.g., a deoxyribonuclease; a DNase).

In another embodiment, a polypeptide (e.g., an antibody) can be conjugated to a "receptor" (e.g., streptavidin) for tumor pre-targeting, wherein the polypeptide receptor conjugate is administered to a patient, followed by removal of unbound conjugate from circulation using a clearing agent, followed by administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide).

In some embodiments, the polypeptide may be conjugated to a prodrug activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic) to an active anticancer drug. The enzyme component of the immunoconjugate comprises any enzyme capable of acting on the prodrug in such a way as to convert it to its more active, cytotoxic form.

Useful enzymes include, but are not limited to, alkaline phosphatase that can be used to convert a phosphate-containing prodrug into the free drug; arylsulfatase useful for converting sulfate-containing prodrugs into free drug; cytosine deaminase for converting non-toxic 5-fluorocytosine to the anticancer drug 5-fluorouracil; proteases such as seratia protease, thermolysin, subtilisin, carboxypeptidase, and cathepsin (e.g., cathepsin B and L), which can be used to convert peptide containing prodrugs into free drugs; a D-alanylcarboxypeptidase useful for converting prodrugs containing D-amino acid substituents; carbohydrate cleaving enzymes, such as beta-galactosidase and neuraminidase, can be used to convert glycosylated prodrugs into free drugs; beta-lactamases useful for converting drugs derivatized with beta-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, may be used to convert drugs derivatized with phenoxyacetyl or phenylacetyl groups, respectively, at their amine nitrogens into free drugs. Alternatively, antibodies with enzymatic activity, also referred to in the art as "abzymes," can be used to convert prodrugs into free active drugs.

(iv) Others

Another covalent modification of a polypeptide includes linking the polypeptide to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol. The polypeptide may also be embedded in microcapsules, such as microcapsules prepared by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly- (methyl-methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences,18th edition, gennaro, A.R., ed., (1990).

Obtaining polypeptides for use in formulations and methods

The polypeptides used in the assays described herein can be obtained using methods well known in the art, including recombinant methods. The following section provides guidance regarding these methods.

(A) Polynucleotide

"polynucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer of nucleotides of any length, including DNA and RNA.

Polynucleotides encoding polypeptides may be obtained from any source, including but not limited to a cDNA library prepared from tissues thought to have polypeptide mRNA and express it at detectable levels. Thus, polynucleotides encoding polypeptides may be conveniently obtained from cDNA libraries prepared from human tissue. The gene encoding the polypeptide may also be obtained from a genomic library or by known synthetic methods (e.g., automated nucleic acid synthesis).

For example, the polynucleotide may encode a complete immunoglobulin molecule chain, such as a light chain or a heavy chain. The complete heavy chain not only includes the heavy chain variable region (V) _H ) Also included is a heavy chain constant region (C) _H ) Which generally comprises three constant domains C _H 1，C _H 2 and C _H 3; and a "hinge" region. In some cases, the presence of a constant region is desirable.

Other polypeptides that can be encoded by a polynucleotide include antibody fragments that bind antigen, such as single domain antibodies ("dAbs"), fv, scFv, fab 'and F (ab') ₂ And "minibodies". The minibody is (usually) excising C _H 1 and C _K Or C _L Bivalent antibody fragments of domains. Since minibodies are smaller than conventional antibodies, they should achieve better tissue penetration in clinical/diagnostic use, but they are bivalent, they should retain higher binding affinity than monovalent antibody fragments (e.g. dabs). Thus, unless the context dictates otherwise, the term "antibody" as used herein includes not only intact antibody molecules, but also antigen-binding antibody fragments of the type described above. Preferably, each framework region present in the encoded polypeptide will comprise at least the corresponding human acceptor frameworkOne amino acid substitution. Thus, for example, a framework region may comprise a total of three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions relative to the acceptor framework region.

Exemplary embodiments

Embodiment 1. In some embodiments, there is provided a method for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein interference between the nonionic surfactant and the polypeptide is reduced during the quantifying, wherein the method comprises the steps of:

a) Applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising mobile phase a and mobile phase B, wherein mobile phase a comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of an acid, wherein the polypeptide binds specifically and non-specifically to the chromatography material;

b) Eluting the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step a);

c) Eluting the non-ionic surfactant and non-specifically bound polypeptide from the chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step c);

d) Quantifying the nonionic surfactant, wherein interference between the nonionic surfactant and the polypeptide during the quantifying is reduced.

Embodiment 2. In some other embodiments of embodiment 1, the ratio of mobile phase B to mobile phase a in step a) is about 10.

Embodiment 3. In some other embodiments of embodiments 1 or 2, the ratio of mobile phase B to mobile phase a is increased to about 40.

Embodiment 4. In some other embodiments of any one of embodiments 1 to 3, the ratio of mobile phase B to mobile phase a is increased to about 100 in step c).

Embodiment 5. In some other embodiments of any one of embodiments 1 to 4, mobile phase a comprises an aqueous solution of about 2% acid.

Embodiment 6. In some other embodiments of any of embodiments 1 to 5, mobile phase B comprises about 2% acid in methanol.

Embodiment 7. In some other embodiments of any one of embodiments 1 to 6, the acid is formic acid.

Embodiment 8. In some other embodiments of any one of embodiments 1 to 6, the acid is acetic acid.

Embodiment 9 in some other embodiments of any one of embodiments 1 to 8, the flow rate for chromatography is about 1.25 mL/min.

Embodiment 10 in some other embodiments of embodiment 9, step b) begins about 1 minute after chromatography begins and ends about 3.4 minutes after chromatography begins.

Embodiment 11. In some other embodiments of 9 or 10, step c) begins about 3.5 minutes after the start of chromatography and ends about 4.6 minutes after the start of chromatography.

Embodiment 12 in some other embodiments of any one of embodiments 1 to 11, the non-ionic surfactant is poloxamer (P188) or a polysorbate.

Embodiment 13 in some other embodiments of embodiment 12, the polysorbate is polysorbate 20 or polysorbate 80.

Embodiment 14. In some other embodiments of any of embodiments 1 to 13, the concentration of the nonionic surfactant in the composition is from about 0.001% to 1.0% (w/v).

Embodiment 15 in some other embodiments of any one of embodiments 1 to 14, the concentration of the protein in the composition is from about 1mg/mL to about 250mg/mL.

Embodiment 16 in some other embodiments of any one of embodiments 1 to 15, the formulation has a pH of about 4.5 to about 7.5.

Embodiment 17. In some other embodiments of any one of embodiments 1 to 16, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers and tonicity agents.

Embodiment 18 in some other embodiments of any one of embodiments 1 to 17, the composition is a pharmaceutical formulation suitable for administration to a subject.

Embodiment 19. In some other embodiments of any one of embodiments 1-18, the polypeptide is a therapeutic polypeptide.

Embodiment 20 in some other embodiments of embodiment 16, the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, thiomab ^TM Or Thiomab ^TM A drug conjugate.

Embodiment 21. In some other embodiments of any one of embodiments 1 to 20, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer.

Embodiment 22. In some other embodiments of any one of embodiments 1 to 21, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety.

Embodiment 23. In some other embodiments of any one of embodiments 1 to 22, the mixed mode anion exchange chromatography material comprises a solid support.

Embodiment 24. In some other embodiments of any one of embodiments 1 to 23, the mixed mode anion exchange chromatography material is contained in a column.

Embodiment 25. In some other embodiments of any one of embodiments 1 to 24, the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material.

Embodiment 26. In some other embodiments of any one of embodiments 1 to 25, the mixed mode anion exchange chromatography material is

MAX chromatography materials.

Embodiment 27. In some other embodiments of any one of embodiments 1 to 26, the nonionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD).

Embodiment 28 in some embodiments, there is provided a method for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the steps of:

a) Applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising mobile phase a and mobile phase B, wherein mobile phase a comprises an aqueous ammonium hydroxide solution and mobile phase B comprises a solution of ammonium hydroxide in an organic solvent;

b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step a);

c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step c);

d) The nonionic surfactant was quantified.

Embodiment 29 in some other embodiments of embodiment 28, the organic solvent of mobile phase B is methanol.

Embodiment 30 in some other embodiments of embodiments 28 or 29, the ratio of mobile phase B to mobile phase a in step a) is about 10.

Embodiment 31 in some other embodiments of any one of embodiments 28 to 30, in step B) the ratio of mobile phase B to mobile phase a is increased to about 45.

Embodiment 32. In some other embodiments of any one of embodiments 28 to 31, in step c) the ratio of mobile phase B to mobile phase a is increased to about 100.

Embodiment 33. In some other embodiments of any one of embodiments 28 to 32, mobile phase a comprises about 2% aqueous ammonium hydroxide.

Embodiment 34 in some other embodiments of any one of embodiments 28 to 33, mobile phase B comprises about 2% ammonium hydroxide in methanol.

Embodiment 35 in some other embodiments of any one of embodiments 28 to 34, the flow rate for chromatography is about 1.4 mL/min.

Embodiment 36. In some other embodiments of embodiment 35, step b) begins about 1 minute after the start of chromatography and ends about 4.4 minutes after the start of chromatography.

Embodiment 37. In some other embodiments of 35 or 36, step c) begins about 4.5 minutes after the start of chromatography and ends about 7.6 minutes after the start of chromatography.

Embodiment 38 in some other embodiments of any one of embodiments 28 to 37, the nonionic surfactant is a polysorbate.

Embodiment 39 in some other embodiments of embodiment 38, the polysorbate is polysorbate 20 or polysorbate 80.

Embodiment 40 in some other embodiments of embodiment 38 or 39, the concentration of polysorbate in the composition is about 0.001% to 1.0% (w/v).

Embodiment 41 in some other embodiments of embodiment 28, the organic solvent of mobile phase B is acetonitrile.

Embodiment 42. In some other embodiments of embodiment 41, the ratio of mobile phase B to mobile phase a in step a) is about 10.

Embodiment 43. In some other embodiments of embodiments 41 or 42, the ratio of mobile phase B to mobile phase a in step B) is increased to about 40.

Embodiment 44. In some other embodiments of any one of embodiments 41 to 43, the ratio of mobile phase B to mobile phase a in step c) is increased to 100.

Embodiment 45 in some other embodiments of any one of embodiments 41 to 44, the mobile phase a comprises about 2% aqueous ammonium hydroxide.

Embodiment 46. In some other embodiments of any one of embodiments 41 to 45, mobile phase B comprises about 2% ammonium hydroxide in acetonitrile.

Embodiment 47. In some other embodiments of any one of embodiments 41 to 46, the nonionic surfactant is a poloxamer.

Embodiment 48 in some other embodiments of embodiment 48, the poloxamer is poloxamer P188.

Embodiment 49 in some other embodiments of embodiment 48 or 49, the concentration of poloxamer in the composition is between about 0.001% and 1.0% (w/v).

Embodiment 50. In some other embodiments of any one of embodiments 28 to 49, the composition further comprises N-acetyltryptophan and/or methionine.

Embodiment 51. In some other embodiments of embodiment 50, the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM.

Embodiment 52 in some other embodiments of embodiment 50, the concentration of methionine in the composition ranges from about 0.1mM to about 100mM.

Embodiment 53. In some other embodiments of any one of embodiments 28 to 52, the concentration of the polypeptide in the composition is from about 1mg/mL to about 250mg/mL.

Embodiment 54 in some other embodiments of any one of embodiments 28 to 53, the formulation has a pH of about 4.5 to about 7.5.

Embodiment 55 in some other embodiments of any one of embodiments 28 to 54, the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers and tonicity agents.

Embodiment 56 in some other embodiments of any one of embodiments 28-55, the composition is a pharmaceutical formulation suitable for administration to a subject.

Embodiment 57 in some other embodiments of any one of embodiments 28-56, the polypeptide is a therapeutic polypeptide.

Embodiment 58. In some other embodiments of embodiment 57, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB ^TM And Thiomab ^TM A drug conjugate.

Embodiment 59 in some other embodiments of any one of embodiments 28 to 58, the mixed mode cation exchange chromatography material comprises a reversed phase strong cation exchange polymer.

Embodiment 60. In some other embodiments of any one of embodiments 28 to 59, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety.

Embodiment 61 in some other embodiments of any one of embodiments 28 to 60, the mixed mode cation exchange chromatography material comprises a solid support.

Embodiment 62. In some other embodiments of any one of embodiments 28 to 61, the mixed mode cation exchange chromatography material is contained in a column.

Embodiment 63. In some other embodiments of any one of embodiments 28 to 62, the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material.

Embodiment 64 in some other embodiments of any one of embodiments 28 to 63, the mixed mode cation exchange chromatography material is

MCX chromatography material.

Embodiment 65. In some other embodiments of any one of embodiments 28 to 64, the non-ionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD).

All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

Further details of the invention are illustrated by the following non-limiting examples. The disclosures of all references in the specification are expressly incorporated herein by reference.

Detailed Description

The following examples are intended to be purely exemplary of the invention and therefore should not be considered as limiting the invention in any way. The following examples and detailed description are provided for the purpose of illustration and not for the purpose of limitation.

Materials and methods of the examples

The following materials and methods were used in the examples unless otherwise indicated.

Material

All mAbs (A1-A20) were prepared using a stable Chinese Hamster Ovary (CHO) cell line or E.coli cells.

Waters

MAX cartridges (2.1x20mm, 30 μm particle size, PN # 186002052) and Waters

MCX cartridges (2.1x20mm, 30 μm particle size, PN # 186002051) were purchased from Waters. Polysorbate 20 was obtained from Sigma (P/N T2700-100 ML). Formic acid was obtained from Fluka (P/N94318-250 ML-F). HPLC grade glacial acetic acid was obtained from JT Baker (P/N9515-03). HPLC grade isopropanol (P/N PX 1834-1) and methanol (P/N MX 0488-1) were obtained from Omnisolv. HPLC grade water was obtained from Honeywell (Cat. No. 365-4). 27-31% ammonia solution was obtained from Spectrum Chemicals (P/N AM 180).

Example 1 optimization

A more robust solution is needed to eliminate protein interference and produce consistent PS20 quantitation across all HPLC column batches. As a starting point, antibody Drug Conjugate (ADC) formulations are used to evaluate the effectiveness of modifications to previous methods before other molecules are evaluated with the methods of the invention. The objective of this experiment was to develop a PS assay to allow robust quantification of PS20 across multiple product formulations by minimizing or completely removing protein interference to demonstrate that the assay of the present invention improves the accuracy and reproducibility of PS20 quantification, including robustness between multiple batches of cartridges, as compared to previous assays, and to conduct qualification studies to assess the accuracy, precision, specificity, reproducibility, and intermediate precision of PS20 in selecting product formulations.

Method

For all experiments, the following ELSD settings were used:

the light source intensity (LED) was set at 75%

Detector gain (PMT) set to 1

In the HPLC assay, the PS20 ester remains in the cartridge, while the other excipients, proteins and non-esterified PS20 species elute in the flow-through or wash steps. After the washing step, an Evaporative Light Scattering Detector (ELSD) was placed in-line using a diverter valve and the esterified polysorbate species were eluted with a step gradient of higher% organic phase. The non-esterified PS20 species account for about 20% of the total PS20 composition (Hewitt, d.et al, 2011, j.chromatography a,1218: 2138-2145). The HPLC-ELSD assay only quantitates the PS20 ester, which is sufficient as long as the PS20 batch used to prepare the standard contains a similar amount of PS20 ester as compared to the PS20 in the sample. Similar PS20 ester compositions of standards can be ensured by equivalent schemes (Hewitt, d.et al, 2011, j. Chromatography a, 1215.

The HPLC-ELSD conditions for the original method (also referred to as method 0) are Agilent 1200 HPLC and Varian 380 ELSD; the cylinder is a Waters Oasis MAX online cylinder; mobile phase a was 2% aqueous formic acid; mobile phase B was 2% formic acid in isopropanol; the flow rate is 1mL/min; the injection volume was 20. Mu.L. The gradient used is shown in table 2.

TABLE 2 method 0 gradient

The following summarizes the modifications of the original method, marked in bold/underline, of the final method, also referred to as method 1. The LC gradient is shown in table 3 and a typical injection sequence for this assay is shown in table 4.

The HPLC-ELSD conditions are Agilent 1200 HPLC and Varian 380 ELSD; the cylinder is a Waters Oasis MAX online cylinder; the mobile phase A is 2% formic acid aqueous solution or 2% acetic acid aqueous solution; mobile phase B was 2% formic acid in isopropanol or 2% acetic acid in methanol; the flow rate is 1.25mL/min; the injection volume was 20 μ L (depending on the PS20 concentration range). Although acetic acid was finally chosen as the final condition, some qualitative and robust work was initially done in the mobile phase using 2% formic acid.

TABLE 3 method 1 gradients

Time (minutes)	% mobile phase A	% of mobile phase B	Description of the steps
				0	90	10	Sample loading
1	60	40	Washing machine
				3.4	60	40	Washing machine
3.5	0	100	Elution is carried out
				4.6	0	100	Elution is carried out
4.7	90	10	Balancing
				6.6	90	10	Balancing

TABLE 4 typical sequence

Method optimization

1. Conditions of initial trial:

during the development of method 1, a number of different experimental conditions were evaluated before selecting the final methanol-based mobile phase in order to minimize the effect of protein interference. These experiments are summarized in table 5.

TABLE 5 brief summary of other attempted solutions (formic acid in mobile phase unless otherwise stated)

Method robustness experiment

Tables 6 and 7 describe the products and cartridges used to test the robustness of the method across multiple products.

TABLE 6 products tested when comparing method accuracy between nine cylinders

TABLE 7 adsorbent batches and batches for Process robustness testing

1. Robustness of the method between columns:

table 8 describes columns for evaluating 12 the robustness of the method with reference to the standard (shown in table 9), using two different columns, one providing acceptable specificity in method 0 and the other providing unacceptable specificity in method 0. The column numbers provided in table 8 correspond to those provided in table 7.

TABLE 8 column for demonstrating process robustness across multiple products

Column body	Performance of	Adsorbent batch	Batch #	HPLC/ELSD system
					1	Acceptable	57	57333161	Agilent/Agilent
5	Is not acceptable	49	49321291	Agilent/Agilent

TABLE 9 reference standard group (20. Mu.L injection)

Method characterization

Characterization of the method was done by assessing linearity, accuracy, precision (including intermediate precision) and specificity. Table 10 describes the instrument and cartridge for intermediate precision.

TABLE 10 conditions for intermediate precision

Sky	System (HPLC/ELSD)	Column number (watch 7)	Analyst
				1	Agilent 1200/Agilent 380	1	1
2	Waters 2695/Varian 380	5	1
				3	Agilent 1200/Agilent 380	7	2

As a result:

method optimization

Multi-step gradient experiments:

during the evaluation of the different mobile phases, a method is needed to determine whether the polysorbate can be completely separated from the other components of a typical protein formulation. To perform this analysis, a multi-step gradient experiment was performed (fig. 1). These experiments required a mobile phase equilibration column consisting of 2% volatile acid (formic, trifluoroacetic or acetic acid) in 10% organic solvent, increasing the concentration of organic solvent in the mobile phase in 5% steps and holding at each concentration for 1 minute to simulate the washing step of the PS20 quantitation method. These 5% steps were repeated until a 98% organic (+ 2% volatile acid) mobile phase was reached. A constant flow rate of 1mL/min was maintained during the process. These experiments were performed with PS20 free protein samples to determine the mobile phase composition of all components of the formulation, except for PS20, completely eluted, as well as the aqueous PS20 standard solution used to determine the mobile phase composition at which to begin elution of the PS20 ester species. The two mobile phase organic solvent concentrations were compared to determine if the optimum mobile phase composition could be found to completely separate the protein matrix component from the polysorbate. This optimal mobile phase composition will be used during the washing step of the analytical method to remove any proteins that may remain in the cartridge. Multistep gradients are more desirable than linear gradients for two reasons. Firstly, the linear gradient does not mimic the washing step of the method, and secondly, it is difficult to determine the discrete mobile phase composition that causes elution of each component during the linear gradient.

Multi-step gradient experiments showed that the optimal methanol concentration for washing proteins from the cartridge but retaining PS20 ester species was between 40% and 50% (fig. 1 and fig. 2). Notably, 2% of the formic acid was still in the mobile phase. Both 40% and 50% methanol wash steps were tested and both eliminated protein interference from the A1 ADC. The PS20 peak area with 40% methanol wash was on average 38% higher than the PS20 peak with 50% methanol wash (n = 8). Therefore, 40% methanol was selected to improve the sensitivity of PS 20. A lower peak area with 50% wash may indicate that some of the less hydrophobic PS20 ester was eluted during the protein wash step, but this possibility was not investigated further.

FIG. 1 shows an example of a typical step gradient overlay (without PS20 product and PS20 standard). The overlay shows that all proteins elute with 40% -50% methanol, and the PS20 ester begins to elute at 50% methanol. Note that the valve between HPLC and ELSD was in the diversion mode within the first minute of the protein sample to avoid saturation of the detector. The multiple peaks shown in the PS20 standard are likely due to different polysorbate species eluting from the cartridge to increase hydrophobicity. The different protein peaks that elute at each step change have not been characterized. This stacking provides a rapid method to find the optimal concentration of methanol to wash the protein from the cartridge while retaining the PS20 ester.

Multiple gradient experiments were repeated using isopropanol to compare the elution of protein and PS20 regions with methanol. Figure 2 shows different multi-step elution profiles of methanol and isopropanol on two different columns, A1 ADC without PS20 and PS20 standards. First cylinder (trace 1) is evaluated; the first pillar normally proceeds through method 0 (trace 1), but the second pillar is not optimal (trace 2). Multi-step gradient experiments with isopropanol showed that the optimum isopropanol concentration in the washing step was 20%. This is consistent with method 0, which uses 20% isopropanol to wash for 1 to 3.4 minutes. However, the separation of protein and PS20 esters from isopropanol washes was inconsistent among different cartridges and demonstrated variability in protein interference from cartridge to cartridge. On some cartridges (e.g., the cartridge used in fig. 2), a portion of the protein eluted at the same isopropanol concentration as the PS20 ester. This behavior suggests that these particular cartridges exhibit significant amounts of protein interference when PS20 is quantitated by the isopropanol method, and that this effect is experimentally confirmed.

In contrast to the lower panel of fig. 2, the upper panel shows that the methanol multi-step gradient over the different columns consistently elutes all proteins through the 40% methanol step, with PS20 esters starting at 50% methanol, indicating a wider separation window for the protein region from the PS20 region compared to the isopropanol step gradient. This result indicates that washing with 40% methanol will consistently separate the protein from the PS20 esters, thereby reducing variability in PS20 quantification between cartridges.

A multi-step gradient experiment was used to rapidly assess the separation of PS20 and protein between different cartridge and mobile phase compositions (fig. 1 and 2).

This method successfully determines the washing conditions of the PS20 assay and can potentially be used to assess the effectiveness of the washing step for separating other analytes using different cartridges and flow phases.

Experiment design:

a stage 2 full-factorial experimental design (DoE) was used to optimize the improved PS20 process with formic acid. The parameters examined were as follows:

methanol concentration in washing step (40%, 50%)

Washing time (1.8 min, 3.0 min)

Flow rate (0.75 mL/min,1.25 mL/min)

Mass of PS20 loaded (4. Mu.g, 12. Mu.g)

Note that the "washing" step refers to a portion of the HPLC method in which the organic concentration is kept constant to wash any residual protein from the cartridge. In addition to the 2-step full-factor arrangement, the midpoint was tested at the beginning and end of the sequence (45% organic wash, 2.4 min wash duration, 1mL/min flow rate, 8 μ g PS20 loading). The experiment was run on three samples, PS20 in water, A1 ADC without PS20 and PS20 incorporated into A1 ADC without PS20, respectively. For the A1 ADC sample without PS20, the PS20 loading parameter does not apply.

The criteria for optimization are as follows:

minimizing protein interference at PS20 retention (only for A1 ADC samples without PS 20)

Maximizing resolution between protein and PS20 peaks (for PS20+ A1 ADC samples)

Minimizing PS20 peak width at 10% peak height (PS 20 in water sample)

Maximize PS20 peak area (PS 20 in water sample)

The results of DoE show that all test conditions eliminate protein interference. No protein interference was observed upon injection of A1 ADC without PS 20. Similarly, the resolution between the protein and PS20 peaks was found to be greater than 3 for all cases, where the resolution was calculated by the following equation (equation 1):

equation 1 resolution

Where t is the retention time of each peak, W _50％ Is the peak width at 50% height.

Since no effect on protein interference or resolution was observed for all test conditions, the PS20 peak area and width were optimized for sensitivity and peak shape. A summary of the effect of flow rate, methanol wash concentration and wash time on PS20 peak area and width is shown in figure 3. Increasing the flow rate reduced the PS20 peak width, and decreasing the methanol concentration from 50% to 40% in the wash step increased the PS20 peak area by 38%. With a 50% methanol wash, additional PS20 peaks eluted in the wash step with retention time later than the non-esterified PS20 species, and this additional peak was suspected to be due to the earlier elution of the less hydrophobic PS20 ester. The most relevant finding was that the PS20 peak area decreased with increasing MeOH concentration in the wash stage and the increasing flow rate decreased the peak width.

A comparison of 40% and 50% methanol washes is shown in figure 4. There was no additional peak present from about 1.8 to 3.5 minutes of washing with 45% methanol. The PS20 loading tested was found to have no effect on any evaluation criteria. The lowest methanol wash concentration (40%), highest flow rate (1.25 mL/min) and mid-wash time (2.4 min) were chosen as the optimization parameters for method 1. The selected final conditions are shown in table 3.

Method robustness experiment

Once the optimal methanol wash concentration was determined using a multi-step gradient experiment, the improvement of method 1 (formic acid + methanol) over method 0 (formic acid + isopropanol) was tested.

Product-to-product process robustness

To further compare the effect of protein interference between method 1 (formic acid + methanol) and method 0 (formic acid + isopropanol), PS20 was quantified using 2 columns in 12 different reference standards. Columns were chosen such that one column (column 1) was considered acceptable, using method 0 yielded accurate results, and one column (column 5) was considered unacceptable due to the high levels of protein interference using method 0. The objective of this experiment was to demonstrate that method 1 reduces the degree of variability between "acceptable" and "unacceptable" cartridges for a variety of products. The consistency of PS20 quantification using each method was compared on the columns and reference standards described in the methods in table 8 and table 9, respectively.

The reference standard for each method and its measured PS20 concentration are listed in table 11. The accuracy of each method was not calculated because the PS20 concentrations listed in C of a cannot be treated as theoretical values for each reference standard, as was done in the case of spiked samples. Therefore, the accuracy of each method cannot be evaluated for these reference standards.

TABLE 11 comparison of methods the column-to-column differences in measured PS20 concentrations for various reference standards

* FA = formic acid

* Calculate absolute% difference in PS20 quantitation for each method by equation 2

The data in table 11 show that the difference in PS20 quantitation between the two columns using methanol in the mobile phase was consistently less than 5%. In contrast, the difference in the quantification of PS20 when isopropanol was used in the mobile phase indicated a much greater degree of variability between columns. Method 0 (formic acid + isopropanol) is more sensitive to column changes. The data in table 11 show that the use of formic acid + methanol in the mobile phase can produce a more consistent PS20 quantification in the different cartridges for all products, regardless of the performance of the cartridges in method 0. The absolute% difference in PS20 quantification was calculated using equation 2:

equation 2 absolute% difference in ps20 quantification

|100 | (concentration of column 5 ] - [ concentration of column 1 ])/[ concentration of column 1 ]/air ventilation

Method robustness between columns

Method 0 and method 1 (formic acid + methanol) were compared by testing two conditions between nine columns with four products. PS20 was incorporated into the protein matrix without PS20 at 50% of the target formulation concentration for each product. This method represents the minimum PS20 concentration required for assay quantification of each product based on analysis of the acceptance criteria certificate. Standards and controls were prepared by incorporating PS20 into water. The tests were performed using two HPLC/ELSD systems (Agilent 1200/Agilent 380 and Waters 2695/Varian 380). The products and cartridges tested are detailed in the methods section, tables 6 and 7, respectively. The tested cartridges were carefully selected to cover a wide range of batches, ages and historical performance.

Samples without PS20 product and PS20 incorporation samples were tested to assess protein interference with PS20 assay and accuracy of PS20 quantification, respectively. A1 ADC, A2, A3 and A4 samples were tested on 9 cartridges with formic acid + methanol and formic acid + isopropanol (method 0). The average recovery, relative standard deviation and range between all columns using both methods are shown in table 12.

TABLE 12 in 9 Waters

Recovery and% RSD of PS20 with protein without PS20 spiked between MAX cartridges.

Table 12 shows that the use of methanol in the mobile phase improves the accuracy of PS20 quantification (average recovery close to 100%) and reduces the variability between columns (% RSD reduction). These data indicate that the use of methanol instead of isopropanol in the mobile phase is particularly suitable for PS20 quantification in A1 ADC and A2 formulations. The biased over-recovery of PS20 in the A4 formulation may be due to a reduced signal-to-noise ratio, loading only 1 μ g of PS20 in the cartridge (20 μ L injection, 0.05mg/mL PS20 concentration). Increased PS20 loading (50 μ L injection) was used during R & D method identification (formic acid + methanol) and no over recovery was observed.

Excessive recovery of A3 was observed when methanol was used in the mobile phase, however, this was still an improvement over the isopropanol mobile phase. It is hypothesized that the low pI of this molecule, which causes retention of the protein by electrostatic attraction on the positively charged quaternary amine groups of the stationary phase, is partly due to the increased abundance of sialic acid groups on its glutathione.

Further method optimization

Selection of mobile phase acid

Experiments were performed using a methanol-based mobile phase and a step gradient of different mobile phase organic acids to determine the optimal additives for the method of minimizing protein interference. Methanol step gradient experiments were performed with ELSD detection using A1 ADC without PS20 and a10 ADC without PS20 using formic acid, acetic acid or trifluoroacetic acid to monitor the effect of acid on protein elution. An aqueous solution of PS20 standard was used to monitor the retention of PS 20.

The mobile phase additive was varied to give an insight into how the protein remained on the cartridge. Figures 5,6 and 7 show the multi-step gradient stacking of methanol for ADC without PS20 and PS20 standards, respectively, using trifluoroacetic acid, formic acid and acetic acid, respectively, in the mobile phase. For all mobile phase additives, PS20 ester began to elute at 50% methanol. Figure 5 shows a PS 20-free a10 ADC and PS20 standards using trifluoroacetic acid in the mobile phase. Protein elution was completed at about 80% methanol. Thus, most of the protein co-elutes with the PS20 ester and interferes with PS20 quantification. Figure 6 shows A1 ADC without PS20 and PS20 standards with formic acid in the mobile phase. The protein was completely eluted at about 40% methanol, indicating that a 40% methanol wash separated the protein from the polysorbate. This result is consistent with method 1 that has been developed. Figure 7 shows A1 ADC without PS20 and PS20 standards with acetic acid in the mobile phase. In this case, the protein was completely eluted with 15% methanol. This finding indicates that proteins can be separated from PS20 esters by any methanol wash in the range of 15% -50%. The ionic pairing strengths of these mobile phase additives are as follows trifluoroacetic acid > formic acid > acetic acid. Accordingly, as the strength of the ion pairing agent in the mobile phase increases, the protein is more strongly retained, as would be expected if the protein were bound to reverse phase immobilization through hydrophobic interactions.

Without being bound by theory, the low pH of the acidic mobile phase creates a net positive charge on the protein. Positively charged proteins and positively charged

The interaction of the MAX stationary phase should be coulombic unfavourable, resulting in the protein not being retained by the stationary phase. In contrast, ion-pairing mobile phase additives can interact with proteins to effectively make them more hydrophobic (Xindu, g.,&regnier, F.E. journal of Chromatography A,296, 1984). This interaction will allow the protein to be retained in the cartridge by a hydrophobic interaction mechanism with a sufficiently strong ion pairing agent (e.g., TFA). Reducing the strength of the ion pairing agent in the mobile phase will subsequently reduce the protein/stationary phase interaction. As the weakest ion pairing agent of the three tests, acetic acid in the mobile phase appeared to significantly reduce protein retention on the cartridge. Thus, acetic acid as a mobile phase additive allowed better separation between the protein and PS20 ester than the other additives tested.

Figure 8 shows a comparison of analytical method performance between acetic acid and formic acid as mobile phase additives. As shown in fig. 8, a very small increase (-3 mV) in baseline for protein injection was observed compared to water injection with formic acid as the mobile phase additive. While this interference is considered minimal, it was observed that protein matrix injection did not increase ELSD baseline when acetic acid was the additive in the mobile phase. In addition, the absorbance at 280nm was monitored for diagnostic purposes to observe protein retention and clearance. When acetic acid was used in the mobile phase, the protein was almost completely eliminated in the empty volume, whereas when formic acid was used as additive the protein was weakly retained on the cartridge and eluted at retention times between 1-2 minutes. Because the LC flow was diverted into the ELSD at 2.4 minutes, the likelihood that the protein would not be completely eliminated when formic acid was the additive is increased, allowing for some protein interference.

Representative chromatograms for different columns using acetic acid + methanol elution conditions (see batch specific details of table 7) are shown in figure 9. Although the peak shape from column 1 is typical (trace 1), these data indicate that the PS20 peak profile can vary from column to column. On some pillars, such as pillar 4 (trace 2), the PS20 peak is tail-biased; with further use of the pillars, the tail may eventually become PS20 peak split (trace 3). This behavior is not common because columns 2 (data not shown) and 4 are the only columns tested, which exhibit peak splitting throughout the process development. In other cases, such as cylinder 5 (trace 4), a slight peak tailing was observed. Incidentally, when isopropanol is used in the mobile phase, the column showing increased peak tailing and peak splitting with the methanol mobile phase is also subject to increased levels of protein interference. This finding may indicate that the pillars showing increased peak tailing retain the hydrophobic molecules more strongly. Regardless of the peak shape, the PS20 quantification and integration processing methods were unaffected.

The final method 1 for PS20 quantification will be carried out with 2% acetic acid in the mobile phase, since it is relative to that of

Formic acid on MAX cartridge was shown to improve the separation of protein and PS20 (fig. 9).

Method of identification

After using a step gradient experiment to optimize the washing step and performing a process robustness experiment, it was found that using 2% acetic acid instead of 2% formic acid in the mobile phase improved the separation of protein and PS20 ester. This example thus describes an identification experiment carried out with a formic acid/methanol mobile phase (from the initial process development) and an acetic acid/methanol mobile phase (final process).

The method used to identify the assay is consistent with the HPLC-ELSD assay in method 0, except for the following parameters:

flow rate =1.25mL/min

Mobile phase a =2% aqueous formic acid or 2% aqueous acetic acid

Mobile phase B =2% formic acid in methanol or 2% acetic acid in methanol

The gradients in table 3 were used.

The injection volume was adjusted according to the concentration of PS20 in the formulation to produce a similar load on the column. See table 13.

The identification studies were performed using A1 ADC without PS20, A1 without PS20, A4 without PS20, A5 without PS20 and a11 without PS 20. A known amount of PS20 was incorporated into each sample to determine the accuracy of the assay. The accuracy, precision, specificity, reproducibility and intermediate precision of the assay (additives used for each product see table 13) were evaluated.

Accuracy and precision:

PS20 (Lot MKBL 2646V) was incorporated into protein samples without PS20 at known concentrations (product dependence; see Table 13) to determine the accuracy of the assay. The experiment was performed using a high concentration PS20 stock solution (25 mg/ml) so that protein dilution by spiking was minimized. For each concentration, PS 20-spiked samples were injected in triplicate, unless otherwise noted. The PS20 recovery was used to determine the accuracy of PS20 quantification. The average recovery range was used to determine precision. Linearity within the specified range was also determined (table 13). Table 13 shows the concentration of PS20 incorporated into each product. Note that 2 ranges of DNIB0600S were used to cover the worst case (i.e., lowest) PS20 concentration (0.4 mg/ml) and higher concentrations (0.7 mg/ml) before phase III formulation lockout. Two separate ranges were used because the ELSD settings needed to be changed to accurately quantify PS20 in the range of 0.2mg/ml to 1mg/ml PS20. For each range, the injection volume was changed, instead of changing the detector settings. After formulation lockout, another evaluation of the final formulation (1.2 mg/mL PS 20) and higher protein concentration (40 mg/mL) was performed using a single range.

TABLE 13 products evaluated during method identification

* Repeated injections at three PS20 concentrations

Each set of standards was made from PS20 aqueous solution. Standards were injected in duplicate prior to protein samples. The test article was bracketed with a water control sample of PS20 every 6 th injection (blacked). PS20 controls were prepared separately from the standard (from PS20 Lot MKBJ 7237V). The standard concentrations were selected to encompass the range of PS20 concentrations incorporated into each protein product without PS 20.

The concentration of the PS20 control was selected based on the likely or actual PS20 concentration of the pharmaceutical formulation for each product. Table 14 shows the concentrations of the standard and control PS20 samples used for the analysis in each concentration range.

TABLE 14 Standard Curve and control PS20 concentration for each product

The accuracy and precision of method 1 was tested for a variety of products. Acceptable accuracy results should indicate an average recovery of 80% -120% per spiked concentration. The results for each product are shown in table 15.

TABLE 15 average% recovery and Range

As shown in table 15, the average recovery of all tested products met the acceptance criteria (80% -120%) at all concentrations, ranging from 82.4% to 114.8%. Thus, the assay demonstrates acceptable accuracy and precision for all products tested within the PS20 range.

The measurement range can also be expressed in terms of PS20 mass (rather than PS20 concentration in the formulation) due to injection volume changes. These results indicate that 1.25 (0.025. Mu.g/. Mu.L. Times.50. Mu.L) to 16. Mu.g (1.60. Mu.g/. Mu.L. Times.10. Mu.L) of PS20 can be loaded onto the cartridge and accurately quantitated.

Linearity

Linearity was assessed by determining Pearson correlation coefficient (r) > 0.99. These values are shown in table 16 for each PS20 concentration range.

Table 16 Pearson correlation coefficients over the range of ps20 concentrations

All values of Pearson correlation coefficient are ≧ 0.99 for the PS20 range tested. Thus, linearity is acceptable for this assay across a variety of products.

Specificity:

specificity of 9 products was confirmed by confirming that injection of formulation buffer without PS20 and products without PS20 would not contribute to the PS20 peak. Injection of formulation buffer and protein sample without PS20 was performed in duplicate. Peak areas in the formulated buffer without PS20 and the protein matrix without PS20 were compared to the standard response at 50% of the target PS20 concentration for each product. The acceptance criterion is met if the peak area in the sample without PS20 is less than or equal to 10% of the peak area in the lowest standard. When some protein interference is visible in the PS20 region, a numerical estimate of specificity is derived using the following equation:

Equation 3 calculation of specificity

Specificity = (area without PS20 protein/area of 50% PS20 specification) = 100

As shown in fig. 10 (using A1 ADC), neither the PS 20-free formulation buffer nor the PS 20-free protein sample interfered with the PS20 peak. Specificity values for all other products are given in table 17. Because the target PS20 concentration was selected for the A1 ADC after the formic acid method identification experiment, the lower target PS20 concentration was used to calculate the specificity of the sample (indicated by the asterisk). Using lower target PS20 concentrations will result in higher specificity values, but all values reported are still well below 10%.

TABLE 17 specificity

N/A protein injection without PS20 was the same as water injection.

Repeatability:

samples containing 0.4mg/mL PS20 spiked into 20mg/mL A1 ADC without PS20 and 0.3mg/mL PS20 spiked into 150mg/mL A14/A15 without PS20 were injected 6 times. The peak areas and corresponding concentrations of these injected PS20 are shown in table 18. The% RSD of area and concentration of repeat injections indicates acceptable injection repeatability.

TABLE 18 injection repeatability

The% RSD for area and concentration of repeat injections demonstrates the reproducibility of the assay.

Intermediate precision:

the intermediate precision (formic acid + methanol only) of the three columns was determined on two different HPLC-ELSD systems with 3 different PS20 standards and buffer formulations, and by 2 different analysts. The average of 2 injections is reported in table 19 for each sample per day. The mean and standard deviation of all injections are reported at the bottom of table 19. The injected samples were 0.4mg/mL PS20 spiked into 20mg/mL A1 ADC without PS20, 0.1mg/mL PS20 spiked into A1 without PS20, and 0.1mg/mL PS20 spiked into A4 without PS20. The conditions for each day are shown in table 10 of the methods section. Intermediate precision was performed using formic acid before acetic acid was finalized as a modifier for method 1. Intermediate precision was repeated in the mobile phase without acetic acid, since all other parameters identified were comparable between the two modifiers and since sample preparation was the same for either case.

The% RSD of all three samples in the 3 day test intermediate precision was less than 10%. These results indicate that the assay is consistent for various cartridges, sample formulations, HPLC-ELSD systems and analysts. It was noted that the concentration at day 2 had a slightly decreasing trend.

TABLE 19 intermediate precision (all experiments with formic acid)

And (4) conclusion:

during the development of the new version of the method, the ELSD method 0 assay was modified as follows:

conversion of mobile phase B from isopropanol to methanol

Additive changed from 2% formic acid to 2% acetic acid

The concentration of the organic matter in the washing step was changed from 20% to 40% in the mobile phase B in 1-3.4 minutes

The flow rate was changed from 1.00mL/min to 1.25mL/min.

Overall, these modifications significantly reduce protein interference and variability from column to column performance. Changing the mobile phase B organics from isopropanol to methanol (fig. 2) improved the separation of protein and PS20 compared to the previous conditions. The results of the methanol/FA and isopropanol/FA comparative experiments (tables 11 and 12) show that method 1 significantly improves PS20 quantitation and cartridge-to-cartridge reproducibility. The use of acetic acid instead of formic acid (fig. 8) as a mobile phase additive further reduced the retention of protein to the cartridge. Furthermore, changing the organic wash step from 20% to 40% significantly reduced protein interference.

The identification of this modified assay for a variety of products indicates that the assay is suitable for quantifying PS20 across a variety of molecular forms, protein concentrations, and PS20 concentrations, thus indicating that method 1 is a candidate for a platform PS20 quantification assay.

Example 2 development of HPLC-ELSD Polysorbate 20 quantitation method for formulations containing N-acetyl tryptophan

In evaluating the above PS20 method (method 1), it was found that N-acetyltryptophan (NAT) significantly interferes with polysorbate 20 (PS 20) when present as an additional excipient in the formulation. Although method 1 of example 1 showed minimal protein interference and reduced variability between cartridges for most of the formulations that had been tested, N-acetyltryptophan remained on the cartridge and eluted at the same retention time as PS20 under the conditions of this method. Without being bound by theory, NAT can remain on the cartridge due to the presence of carboxylate groups on the molecule that interact with the mixed mode anion exchange resin (MAX) cartridge used in method 1.

Method 1 is modified to eliminate NAT interference as follows:

1) Change the cartridge from MAX resin to Mixed mode cation exchange resin (MCX)

2) The mobile phase additive was changed from acetic acid to ammonium hydroxide.

Furthermore, further optimization of the method, also referred to as method 2, was performed by increasing the organic wash from 40% b to 45% b and increasing the wash step time and the elution step time by +1 minute and +2 minutes, respectively. The development of conditions for running PS20 quantification for projects formulated using NAT is described in this example. By assessing the accuracy, precision, linearity, specificity, reproducibility and robustness of method 2, three products containing NAT in the formulation were used to assess new conditions.

Higher protein interference with low pI molecules was previously observed with LC-ELSD assays using MAX cartridges. Therefore, the evaluation of low pI molecules was performed on method 1 and method 2 to determine which method is more suitable for correct PS20 quantification.

Material

HPLC/ELSD System, e.g., agilent 1200 HPLC-Varian 380 ELSD

Polysorbate 20 Sigma P/N T2700-100ML

HPLC grade glacial acetic acid JT Baker

Strong ammonia solution, 27-31%: spectrum Chemicals

HPLC grade water Honeywell

HPLC grade methanol Omnisolv

MAX column 2.1x20mm, particle size 30 μm

MCX cylinder 2.1x20mm, particle size 30 μm (Table 20)

NAT containing products (Table 21)

TABLE 20 MCX cartridges used during development of method 2

TABLE 21 product information

The method comprises the following steps:

For all experiments, the ELSD light source intensity (LED) was set to 75% and the detector gain (PMT) was set to 1. The final modifications of the method used to make the assay compatible with NAT-containing formulations are summarized below:

agilent 1200 HPLC (or equivalent) and Varian 380 ELSD (or equivalent)

Column:

MCX on-line cylinder

Mobile phase A1.5% ammonium hydroxide aqueous solution

Mobile phase B1.5% ammonium hydroxide solution in methanol

Flow rate 1.40mL/min

Steering valve timing flow to ELSD at 4.00 min

Injection volume 25. Mu.L (product dependent)

TABLE 22 modified PS20 method gradient (method 2)

As a result:

determination of interference

The initial focus of method development is to determine whether NAT is the source of interference observed in previous assays. This work was done by studying a series of buffers that contained NAT or excluded it (details of buffers provided in the materials section (table 21) and results section (table 23)).

At the target concentration (nominal =80 mg/mL), the a16/a17 formulation buffer without PS20 (20 mM histidine-HCl, 1mM nat,5mM methionine, 240mM sucrose, ph 5.5) and a16/a17 without PS20 were initially evaluated using method 1 of example 1. The composition of each sample is provided below:

formulation buffer without PS 20- -20mM histidine-HCl, 1mM NAT,5mM methionine, 240mM sucrose, pH5.5.

A16/A17 sample without PS 20-80 mg/mL (nominal) in 20mM histidine-HCl, 1mM NAT,5mM methionine, 240mM sucrose, pH 5.5.

Figure 11A shows ELSD results for this evaluation, where significant interference was observed in the ELSD at the PS20 retention times (-4.5 minutes) for both samples. Water (trace 1), formulation buffer without PS20 (trace 2) and protein sample without PS20 (trace 3) injections are shown. In addition, fig. 11B shows the UV (280 nm) signal of each sample, in which the components were detected at the approximate retention time of PS 20. Since the degree of interference observed with the injection of the formulation without PS20 was about the same as with the protein sample, it is clear that the source cannot be entirely attributed to the protein and that we hypothesized that one of the excipients in the buffer was retained by the cartridge.

We determined that n-acetyltryptophan (NAT) is the observed interferer in FIGS. 11A and 11B, because 1) NAT-containing preparations were not previously tested with method 1, and therefore are very different from previous evaluations; 2) NAT has an apparent pKa of 4.1 and has carboxylic acid groups, which make it possible to retain it in the ammonium cation resin of the MAX cartridge in the form of deprotonated anions; 3) The NAT absorbance maximum was close to 280nm (H.Edelhoch, biochemistry, vol.6, no.7, july 1967), and absorbance at 280nm was observed at PS20 retention time (. About.4.3 min) using NAT formulation samples (FIG. 11B).

To confirm NAT interference with PS20, a series of a16/a17 formulation buffers with or without NAT were tested as described above. Fig. 12A and 12B show the HPLC-ELSD results of this evaluation, with the NAT-containing buffers shown in fig. 12A and those without NAT shown in fig. 12B. Each NAT-containing buffer in fig. 12A also shows a peak at the retention time of PS 20. In contrast, all buffers excluding NAT showed no interfering peaks. In summary, the data shown in FIGS. 11A,11B,12A and 12B indicate that NAT remains

MAX cartridge and coeluted with PS20, but not interference caused by proteins or other excipients.

TABLE 23 buffers tested using methods 1 and 2

NAT contains carboxylic acid groups and can be retained in the resin found in the cartridge by anion exchange. We explored the use of alternatives

Column (a)

MCX) toBetter separating NAT from PS 20. With cationic resins containing ammonium groups

The MAX cylinders are different from each other in that,

the MCX cartridge contains an anionic sulfite resin. Initially, the MCX cartridge was evaluated using the same mobile phase (methanol + acetic acid) used in method 1. However, under these conditions, protein interference was found to rise to unacceptable levels (data not shown).

The Sample Extraction method suggests the use of ammonium hydroxide as a mobile phase additive in the solid phase Extraction procedure in the MCX resin plate format (Waters, "Oasis Sample Extraction Products," 2011). Therefore, the mobile phase was modified by replacing 2% acetic acid with 1.5% ammonium hydroxide to better mimic the conditions recommended by the manufacturer. With these method modifications, different derivatives of the a16/a17 buffer were evaluated using MCX cartridges, and the data are shown in fig. 13A and 13B.

Unlike the results obtained with method 1, no interference was observed with the retention time of PS20 with or without NAT and all excipients eluted from the column before 1 min (fig. 13A and 13B). In addition, when 50 μ L of a16/a17 protein without PS20 was injected (fig. 13A, trace 4), no interference was observed in the PS20 region in ELSD, indicating that this method has the potential to separate protein from PS 20. The MCX cartridge and ammonium hydroxide as mobile phase additive were selected for evaluation of PS20 quantification of NAT-containing formulations.

Method modification

Once the preliminary conditions for analyzing NAT-containing formulations are established, the following parameters are examined in further detail, as described above:

ammonium hydroxide in the mobile phase%

% B used in the washing step (20-60% of the test B)

Wash time (2.4 min and 3.4 min) and injection volume (25 and 50 μ L injection volume)

Flow rate (0.8 mL/min to 1.6 mL/min)

Elution time (1.1 min, 3.1 min)

Ammonium hydroxide in the mobile phase%

The Sample Extraction method suggests the use of ammonium hydroxide as a mobile phase additive in a solid phase Extraction procedure in the form of MCX resin plates (Waters, "Oasis Sample Extraction Products," 2011). The experiment was performed by ELSD (fig. 14A) and UV (fig. 14B) detection. The a16/a17 protein without PS20 was the sample used to evaluate protein and NAT elution from the column in the mobile phase using 0.15,0.29,0.73 or 1.5% ammonium hydroxide (fig. 14A and 14B, traces 1,2,3 and 4, respectively). Since the UV detector is on-line before the diverter valve, analytes with chromophores (e.g. NAT and proteins) that elute before PS20 can be detected. To evaluate the ability to clear NAT, a16/a17 formulation buffer without PS20 was also injected (fig. 14A and 14B, trace 5).

When the column effluent was introduced into the ELSD at 2.4 minutes by switching the diverter valve, a slight disturbance of 0.15% ammonium hydroxide (fig. 14A, trace 1) was observed in the mobile phase, as evidenced by a slightly higher baseline; UV traces also indicate that protein and/or NAT are mainly eluted from the column before the effluent enters the ELSD. Most of these potential interferents elute in the empty volume. As the percentage of ammonium hydroxide additive increased (from 0.29% to 1.5%), the difference in the degree of protein or NAT interference observed by ELSD was minimal (fig. 14A, traces 2-4). In addition, the UV trace shows the method of adequate protein and NAT clearance at all tested ammonium hydroxide levels (fig. 14B, traces 2-4). When protein without PS20 was injected with 1.5% ammonium hydroxide in the mobile phase (fig. 14A, trace 4), there appeared to be less protein interference when the effluent was introduced at 2.4 minutes (4.0-5.5 minutes) in the PS20 zone than when the same protein with a lower percentage of ammonium hydroxide was injected. Formulation buffer without PS20 (fig. 14B trace 5) UV trace shows effective NAT scavenging with 1.5% ammonium hydroxide in mobile phase. Therefore, this percentage of additive was selected for the process.

The absorbance at 280nm (fig. 14B) shows that protein and NAT are sufficiently cleared when 0.15-1.5% ammonium hydroxide is used in the mobile phase, and there is some tailing of these interferents at about 2.5 minutes. Because of this finding, the methanol + ammonium hydroxide method was modified to divert the flow to the ELSD at 3.0 minutes instead of 2.4 minutes to prevent detector contamination and to ensure minimal protein and NAT interference in the ELSD. Since the elution of PS20 is at about 4.5 minutes, this change should not affect the peak.

% B used in washing step (20-60% tested)

Previously, to develop method 1 in formulations without NAT, the% methanol used to separate the protein from PS20 during the washing step was optimized using a step gradient method. The% methanol used in the development of method 2 was reconsidered due to the unknown effect on protein retention of several key changes to the method; the stationary phase was changed to MCX resin and the mobile phase additive was changed to ammonium hydroxide. In this experiment, four different levels of% B (methanol +1.5% ammonium hydroxide) in 10% (v/v) increments were tested over a concentration range of 20-60%. Both ELSD and UV detection were used to monitor protein elution for each test condition. Polysorbate-free a16/a17 samples were used for evaluation, and ELSD and UV data are shown in fig. 15A and 15B, respectively.

In fig. 15B, the 280nm absorbance chromatogram shows a clear peak at the retention time of PS2- (-4.5 min), indicating that there is protein and/or NAT retention on the column with a 20-th B-wash (trace 1) when 15 μ Ι _ a16/a17 protein without PS20 was injected. Larger peaks were also observed in the empty volume, which could be a mixture of protein and NAT. For the same conditions, well-defined peaks were also detected in ELSD as interference in the PS20 region (fig. 15A). NAT and protein clearance are improved when the% B of the washing step is increased to 30% (trace 2), as evidenced by the peak reduction of the PS20 region in the UV and ELSD traces. Remove NAT and protein more efficiently with 40% B, but there was still slight interference of the PS20 region in ELSD (FIG. 15A). Washing with 50% and 60% b (traces 4 and 5, respectively) most effectively cleared NAT and protein with minimal interference. 45% in the selection washing step B was used in the method. During the previous development of method 1, we also tested up to 50% methanol as washing conditions, but observed that a small fraction of PS20 would elute earlier under these conditions (this small peak was not observed at 45% methanol). These peaks are likely shorter chain length esters, which are less hydrophobic and indicate that the 50% methanol condition is approximately the point at which the esterified species will elute. Due to this previous observation, and the present results show that 40%B is sufficient to remove the proteins, the 45%conditions, which we decided the washing steps, provide a compromise between robust protein removal and PS20 retention. Furthermore, the UV data in figure 15B shows that the proteins elute slightly earlier under 50% B wash conditions.

Wash time (2.4 min and 3.4 min) and injection volume (25 and 50 μ L injection volume)

To further reduce interference in the PS20 region, wash steps of 2.4 min and 3.4 min were evaluated. As shown in fig. 16, the PS20 region was similar between 2.4 or 3.4 minute washes (trace 3 and trace 2, respectively). Since the specificity was slightly improved and there was no significant negative effect at longer wash times, a wash of 3.4 minutes was chosen for this method. In addition, the flow rate was selected to be diverted to ELSD at 4.0 minutes instead of 2.4 minutes.

The effect of injection volume on baseline was also assessed by reducing the sample volume from 50 μ Ι _ to 25 μ Ι _ (fig. 16, trace 2 and trace 1, respectively). The baseline of the 25 μ Ι _ injection trace appeared slightly clearer, which is expected because less protein and excipients were removed from the column. Finally, the injection volume does not significantly affect performance in terms of specificity.

Flow rate (0.8 mL/min to 1.6 mL/min)

The flow rate was evaluated to ensure that the protein and NAT were effectively cleared. The flow rate was varied between 0.8-1.6 mL/min. FIGS. 17A and 17B show injection of 25 μ L of a sample of A18/A19 protein without PS20 at 150mg/mL using flow rates of 1.60,1.40,1.25,1.00 and 0.80mL/min (traces 1,2,3,4 and 5, respectively).

At the slowest flow rate of 0.8mL/min (trace 5), there was still protein and excipient elution in the PS20 region, as shown by the peaks in the ELSD (fig. 17A). The UV trace of this condition (fig. 17B) also shows increased interferents eluting later than higher flow rates. The interference is reduced when the flow rate is increased to 1.00mL/min (trace 4) and is almost negligible once the flow rate is increased to 1.25mL/min (trace 3). When the flow rate was increased to 1.40 and 1.60mL/min (traces 2 and 1, respectively), the UV trace showed that most of the proteins and excipients were more completely eluted from the cartridge by 2.0 minutes than at the lower flow rate, and a flow rate of 1.40mL/min was selected for the method. We did not increase to 1.6mL/min because the performance was almost equivalent at 1.4mL/min, and because we wanted to minimize the risk of leakage due to the higher back pressure of 1.6 mL/min.

Elution time (1.1 min, 3.1 min)

The elution step of 100% B in method 1 was kept for 1.1 min. A peak of unknown identity eluting at-6.5 minutes was observed at each injection, independent of sample, and including a water blank. The unknown peaks appeared to elute after the mobile phase% B returned to re-equilibrium conditions. To test whether the peak was able to better separate from PS20, the elution time was extended. Fig. 18 shows the integration of the PS20 peak, which starts at 5.2 minutes and ends approximately when the unknown peak starts (trace 2). Although this did not significantly affect quantitation, to prevent potential interference from this peak in the future, the elution step was increased to a retention time of 3.1 minutes (trace 1), allowing for better separation and integration of the PS20 region. FIG. 19 shows the results of using method 2 with the finalized parameters, water, 150mg/mL A18/A19 without PS20, 0.2mg/mL PS20 doped with water, and 0.2mg/mL PS20 doped with A18/A19 ( traces 1,2,3, and 4, respectively).

Method qualification test

Specificity of

Specificity was determined by confirming that injection of formulation buffer without PS20 and products without PS20 did not contribute to any peak that might interfere with the PS20 region; these were compared to the lowest PS20 calibration standard, which was typically 50% of the target PS20 concentration.

Typical chromatograms of formulation buffer without PS20, protein without PS20, and 0.1mg/mL PS20 (50% target) spiked into water are shown in FIGS. 20A-20F. Three different products without PS20 were evaluated. A18/A19 (FIGS. 20A and 20B), A16/A17 (FIGS. 20C and 20D) and A14/A20 (FIGS. 20E and 20F) had protein concentrations of 150,80 and 150mg/mL, respectively. More details on these products are provided in table 21. Visually, all products had some interference in the PS20 region in the ELSD when the protein sample without PS20 was injected (fig. 20a,20c and 20E, trace 2). However, the formulation buffer without PS20 showed no visual interference in the PS20 region (fig. 20A,20C and 20E, trace 1), indicating that the interference is mainly from proteins.

The acceptance criterion of the method is met if the peak area in the PS20 free sample is less than or equal to 10% of the peak area in the minimum criterion. Since some protein interference is visible in the PS20 region, the specificity must be determined using an equation. There are different methods for determining specificity. In this study, specific numerical estimates were derived using the following equation:

Equation 1 specificity

Interference (%) = (area not containing PS20 protein/area of PS20 standard of 50%) + 100

Using this equation, the% interference for the three products was calculated (table 24). A16/A17 without PS20 (80 mg/mL) (FIG. 20C) was determined to have a specificity of 7% compared to 0.1mg/mL of PS20 in water (50% target). A18/A19 without PS20 (150 mg/mL) (FIG. 20A) had only 4% interference, while A14/A20 (150 mg/mL) (FIG. 20E) had 13% interference compared to 0.1mg/mL PS20 in water (50% target).

For all products, UV traces at 280nm (fig. 20b,20d and 20F) show that protein and NAT are effectively cleared when the 4.0 min flow is diverted into ELSD. Although A14/A20 has 13% interference, repeatability, accuracy and linearity are acceptable in subsequent evaluations of the product.

TABLE 24 specificity of the three products

Accuracy of

To determine the accuracy of the assay, a known amount of PS20 was incorporated into the protein without PS20 and the recovery was determined for each concentration (table 25). Typical validation acceptance criteria require% recovery to be within 80-120%.

A18/A19 without PS20 (150 mg/mL) and A16/A17 without PS20 (80 mg/mL) were evaluated by incorporation of PS20 in the range of 0.1-0.6 mg/mL. The accuracy data are summarized in table 25. The recovery ranges for A16/A17 and A18/A19 were 94-100% and 78-100% at the tested PS20 concentrations, respectively. The sample with 78% recovery occurred with 0.4mg/mL PS20 spiked into A18/A19 without PS 20. The recovery in the bracket (scraping) PS20 concentration (0.2 and 0.6 mg/mL) was within specification, indicating sample preparation or injection error for this one sample. If 0.4mg/mL of sample is excluded, the recovery of A18/A19 ranges from 88 to 100%.

The accuracy of A14/A20 without PS20 (150 mg/mL) was assessed by incorporation of PS20 in the range of 0.1-0.3 mg/mL. The PS20 recovery of A20/A20 was 92-109%. Overall, the spiked recovery experimental data for all three tested products indicate that the method is accurate. Furthermore, these results indicate that 2.5 μ g (0.1 μ g/. Mu.L.25 μ L) to 15 μ g (0.6 μ g/. Mu.L.25 μ L) of PS20 can be loaded onto the cartridge and accurately quantified.

TABLE 25 accuracy

Degree of linearity

Linearity was assessed by determining Pearson correlation coefficient (r) ≧ 0.99 over the range of the three product test. These values for each PS20 concentration range are shown in table 26 for each product. All values of Pearson correlation coefficient were greater than 0.99 for the tested PS20 range. Thus, the linearity of the assay was acceptable in the three products tested.

TABLE 26 linearity Pearson correlationNumber of

Product(s)	PS20 concentration Range (mg/ml)	Pearson correlation coefficient (r)
			A18/A19	0.1–0.6	0.9915
A16/A17	0.1–0.6	0.9999
			A14/A20	0.1–0.3	0.9994

Repeatability

Repeatability was assessed by six repeated injections of A18/A19 samples (nominally 0.2mg/mL PS 20) and six repeated injections of A16/A17 samples (nominally 0.2mg/mL PS 20) and measuring% RSD of PS20 peak area. The results of this evaluation are shown in table 27 and show that the accuracy of the assay is acceptable for both products.

TABLE 27 repeatability of A18/A19 and A16/A17

Repeatability was also assessed by testing three replicates of five concentrations and measuring% RSD of PS20 peak area. The experiment was performed with 0.10-0.30mg/mL PS20 spiked into A14/A20 without PS 20. The results of this evaluation are shown in Table 28, and show that the accuracy of this determination is acceptable for A14/A20.

TABLE 28 repeatability of A14/A20

Method robustness experiment

Cylinder to cylinder

Previous work done with method 1 of example 1 has shown that even when the adsorbent batches of the cartridge are the same, the cartridge may exhibit different behavior in terms of protein clearance and specificity. Three MCX cylinders were evaluated to determine cylinder-to-cylinder variability. Wherein MCX columns 2 and 4 have the same adsorbent batch (0093) and 6 is different (0103) (table 20).

All three MCX cartridges were evaluated with 0.2mg/mL PS20 and PS20 containing product spiked into water. The chromatograms for these evaluations are shown in fig. 21 and 22, and a summary of the results is shown in table 29. Traces 3 and 4 in fig. 21 and trace 3 in fig. 22 show the difference in peak height and width of 0.2mg/mL PS20 in water samples injected in the three columns. Most notably, peak heights and areas were variable from pillar to pillar, with the greatest differences observed between pillars 2 (traces 1 and 3 of fig. 21) and pillars 6 (traces 2 and 4 of fig. 21). In both sample types tested (i.e., aqueous solution of PS20, PS20 in A18/A19), the area of the peak using column 2 was about 60% of the area of the peak using column 6. Given that the differences in peak areas observed are independent of sample type (e.g., with or without protein), variability is unlikely to be caused by protein interference. Although the area count difference between the two columns tested was large, the difference between columns 4 and 6 was small, with column 4 having an area of approximately 85% of that of column 6. The variability of the PS20 peak area between columns does not appear to be completely dependent on the resin batch, as columns 2 and 4 share the same resin batch and also produce different peak areas.

Although this variability is not ideal, once the standard is implemented to obtain the calculated amount of PS20, the concentration of PS20 determined from the calibration curve analyzed on the same column is accurate compared to the theoretical concentration in all columns. Based on these data, it is important to utilize the injected volume of PS20, which provides a response well within the linear range of the ELSD detector. For example, for the HPLC-ELSD method, a maximum detector response of 80% of full scale (e.g., 800 mV) is generally recommended, but given the variability observed from column to column, it may be recommended to reduce this target response of the MCX method to further ensure that the detector does not saturate some columns. Overall, the column-to-column variability is minimal relative to the amount of PS20 reported by the method for the samples and columns tested.

TABLE 29 cylinders and variability of cylinders

100X injection of A18/A19 samples

Although the above experiments prove that

MCX cartridges can quantify PS20 robustly and accurately, but we wish to test the durability of the cartridge by running long sequences with protein-containing samples. One of the products originally developed by this method had a very high target protein concentration of 150mg/mL and could lead to cartridge failure/overpressure due to protein accumulation on the cartridge. Typically, the process is run at a pressure of from-25 to 45 bar. If the pressure increases beyond this range, the cartridge may accumulate proteins and/or excipients and the cartridge should be closely monitored or replaced. If the column begins to leak, it should be discarded and replaced immediately.

The reproducibility of the columns and the ability to continuously clear protein and NAT were assessed by injecting the protein (150 mg/mL) one hundred times with the new columns (see table 30 for sequence). Controls (n = 11) were bracketed every tenth protein injection and analyzed first to ensure that the sequence was valid. FIG. 23 shows the overlay of the control. Although there was an increase at the beginning of baseline (4.0-5.0 min, after 8.5 min) and at the end of baseline (up to 5 mV), these changes did not affect the integration or final PS20 quantification (table 31), and the average PS20 amount was calculated as 0.2 ± 0.005 (2.8% rsd) mg/mL.

TABLE 30 sequences (all 25. Mu.L injections)

Table 31 quantification of controls used in 100x protein sequences

Since the control was qualitatively and quantitatively consistent throughout the run, 100A 18/A19 samples (150 mg/mL) with a nominal 0.2mg/mL PS20 (FIG. 24) were integrated and quantified (FIG. 26B, table 32). FIG. 24 shows the same trend as the control, with increasing baselines (-5 mV) before the PS20 region, at 4.0-5.0 minutes and after 8.5 minutes. Plotting area versus number of injections and number versus number of injections, there was minimal deviation in protein quantification, although the area and number of the first 20 injections tended to decline slightly, probably due to equilibrium of the columns (fig. 26A and 26B). Finally, the average area and amount of PS20 quantified for 100 injections of the a18/a19 sample were 18.3 ± 0.76 (4.2% rsd) mV min and 0.2 ± 0.005 (2.6% rsd) mg/mL, respectively (table 32). Note that 375mg of protein was loaded onto the cartridge by the 100 th injection. This method was sufficient to remove protein and excipients as shown in the UV trace (fig. 25B) until the effluent entered the ELSD at 4.0 minutes throughout the sequence (fig. 25A).

Due to the declining trend, 100 injections of a18/a19 formulation buffer with a nominal 0.2mg/mL PS20 were evaluated using a new MCX column (column 5) to determine whether the effect was due to protein or formulation or both. Interestingly, there was a similar downward trend in area counts [ n =100, mean 31.3 ± 1.3 (4.1% rsd) mv min ] for the first 49 injections (fig. 27A). This behavior had no major impact on quantification [ n =100, mean 0.22 ± 0.005 (2.1% rsd) ] (fig. 27B and table 32).

This does not decrease when 0.2mg/mL PS20 spiked into water is evaluated on a completely new cartridge (cartridge 3). For this sample, the area count [ n =100, average 20.7 ± 0.3 (1.5% RSD) mv × min ] (fig. 28A) remained consistent while quantifying [ n =100, average 0.20 ± 0.002 (0.9% RSD)) ] (fig. 28B, table 32). This result from the water + PS20 sample may indicate that the previously observed downward trend (fig. 26A and 27A) is related to the presence of other excipients or proteins in the formulation. Another possibility is that it is an effect related to the specific cartridge used and is independent of other formulation components.

TABLE 32 quantification of PS20, A18/A19 formulation buffer, A18/A19 samples spiked in water injected 100X

Overall, three columns performed effectively after 100 PS20 injections, demonstrating the reproducibility and durability of MCX columns suitable for routine use in QC environments. More importantly, the absorbance curves of 100 protein injections clearly showed similar clearance of protein and NAT throughout the sequence (fig. 25B). PS20 quantification remained statistically constant throughout the sequence.

Quantitative evaluation of three Low pI products by PS20

Three different low pI products, a21, a14/a15 and a14 (table 33), were evaluated using method 1 (described in example 1) and method 2. The cartridges used for this evaluation are listed in table 34. The performance characteristics of the test method are specificity, accuracy, linearity and repeatability.

TABLE 33 molecules evaluated

* pI was determined by the bracket (calibration curve) method

* pI determined by iciEF control System assay

TABLE 34 cylinders used during development of the method

Specificity of

The calculated specificities for the three molecules are shown in table 35. The corresponding chromatograms are shown in FIGS. 29A-29F. A21 (150 mg/mL) was used in method 1 (containing methanol and acetic acid) as compared to 0.1mg/mL PS20 (50% target) in water

MAX) was evaluated with 16% interference when using method 2 (with methanol and ammonium hydroxide)

MCX) had 7% interference. This is probably due to the weak interaction of the low pI product with the cation exchange resin (sulfite anion groups) minimizing protein interference. Compared to 0.15mg/mL PS20 in water (50% target), a14/a15 (192 mg/mL) had 3% interference when evaluated by method 1 and 2% interference when evaluated by method 2. A14 (161 mg/mL) had about 1% interference when evaluated with both methods compared to 0.15mg/mL PS20 (50% target) in water. The specificity of these products is not significantly affected by the method.

TABLE 35 specificity of three Low pI molecules

Method

1	Method 2
		Product(s)	Specificity (%)	Specificity (%)
A21	15.6				7.1
		A14/A15	3.4	1.9
A14	0.8				0.6

Accuracy of

To determine the accuracy of the assay, known amounts of PS20 were incorporated into proteins without PS20, and the recovery was determined for each concentration (table 36). Typical validation acceptance criteria require% recovery to be within 80-120%.

A21 without PS20 was evaluated by incorporation of PS20 in the range of 0.10-0.30 mg/mL. The accuracy data is summarized in table 36. At the tested PS20 concentrations, the recovery ranged from 99-108% and 101-110% when the samples were analyzed using method 1 and method 2, respectively. The accuracy of A14/A15 and A14 without PS20 was assessed by incorporation of PS20 in the range of 0.15-0.45 mg/mL. When the samples were analyzed using method 1 and method 2, the PS20 recovery of A14/A15 ranged from 97-101% and 92-99%, respectively. When samples were analyzed using method 1 and method 2, the PS20 recovery for A14 was 102-108% and 102-110%, respectively. Overall, the data from the spiking recovery experiments for all three products tested demonstrate that both methods are accurate.

TABLE 36 recovery of the three low pI products

In triplicate, 20. Mu.L injections

Linearity

Linearity was assessed by determining a Pearson correlation coefficient (r) ≧ 0.99 within the test range for the three low pI products assessed using methods 1 and 2. These values are shown in table 37 for each PS20 concentration range for each product. All values of Pearson correlation coefficient were greater than 0.99 for the tested PS20 range. Thus, of the three products tested, linearity was acceptable for method 1 and method 2.

TABLE 37 linearity of three low pI products

Repeatability

Reproducibility was assessed by testing three replicates of five concentrations and measuring the% RSD of the PS20 peak area. The experiment was performed with 0.10-0.30mg/mL of PS20 spiked with A21 without PS20 and 0.15-0.45mg/mL of PS20 spiked with A14/A15 and A14 without PS 20. The results of this evaluation are shown in table 38 and show that the precision of the assay is acceptable for both method 1 and method 2 among the three products tested.

TABLE 38 reproducibility of the three low pI products

Triplicates, 20. Mu.L injections

And (4) conclusion:

the current ELSD assay was modified from method 1 of example 1 as follows:

the method 2 comprises the following steps:

mobile phase additive conversion from 2% acetic acid to 1.5% ammonium hydroxide

The flow rate was changed from 1.25mL/min to 1.40mL/min

The LC flow from the waste into the ELSD was changed from 2.4 minutes to 4.0 minutes

The concentration of B% in the washing step was changed from 40% to 45% mobile phase B

The time for the organic material in the washing step is changed from 1.0-3.4 minutes to 1.0-4.4 minutes

The time of the elution step was changed from 3.5-4.6 minutes to 4.5-7.6 minutes

The time for the equilibration step was changed from 4.7-6.6 minutes to 7.7-9.6 minutes

These modifications eliminate NAT and protein interference and have minimal cylinder-to-cylinder quantitative variability. The results of the identification of the modified assay for the three NAT-containing products indicate that the assay is suitable for quantifying polysorbate 20 in these formulations.

Three low pI molecules were evaluated using methods 1 and 2. When using methods 1 and a21, the only example that does not meet the acceptance criteria is specificity. In addition, both methods passed the accuracy, linearity and reproducibility criteria of all three low pI products. These results further indicate that method 2 is also capable of quantifying PS20 and is particularly useful for NAT-free products.

Detailed description of the preferred embodiments

1. A method for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein interference between the nonionic surfactant and the polypeptide is reduced during the quantifying, wherein the method comprises the steps of:

a) Applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising mobile phase a and mobile phase B, wherein mobile phase a comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of an acid, wherein the polypeptide binds specifically and non-specifically to the chromatography material;

b) Eluting the specifically bound polypeptide from the mixed mode anion exchange chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step a);

c) Eluting the non-ionic surfactant and non-specifically bound polypeptide from the chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step c);

d) Quantifying the non-ionic surfactant, wherein interference between the non-ionic surfactant and the polypeptide is reduced during the quantifying.

2. The method of embodiment 1, wherein the ratio of mobile phase B to mobile phase a in step a) is about 10.

3. The method of embodiment 1 or 2, wherein the ratio of mobile phase B to mobile phase a in step B) is increased to about 40.

4. The method according to any of embodiments 1 to 3, wherein the ratio of mobile phase B to mobile phase a is increased to about 100 in step c).

5. The method of any of embodiments 1-4, wherein mobile phase a comprises an aqueous solution of about 2% acid.

6. The method of any of embodiments 1-5, wherein mobile phase B comprises about 2% acid in methanol.

7. The method of any of embodiments 1-6, wherein the acid is formic acid.

8. The method of any of embodiments 1-6, wherein the acid is acetic acid.

9. The method of any one of embodiments 1-8, wherein the flow rate for chromatography is about 1.25 mL/min.

10. The method of embodiment 9, wherein step b) begins about 1 minute after chromatography begins and ends about 3.4 minutes after chromatography begins.

11. The method of embodiment 9 or 10, wherein step c) begins about 3.5 minutes after the start of chromatography and ends about 4.6 minutes after the start of chromatography.

12. The method of any one of embodiments 1-11, wherein the nonionic surfactant is poloxamer (P188) or a polysorbate.

13. The method of embodiment 12, wherein the polysorbate is polysorbate 20 or polysorbate 80.

14. The method of any of embodiments 1-13, wherein the concentration of the nonionic surfactant in the composition is in the range of about 0.001% to 1.0% (w/v).

15. The method of any one of embodiments 1-14, wherein the concentration of protein in the composition is from about 1mg/mL to about 250mg/mL.

16. The method of any of embodiments 1-15, wherein the formulation has a pH of about 4.5 to about 7.5.

17. The method of any of embodiments 1-16, wherein the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents.

18. The method of any one of embodiments 1-17, wherein the composition is a pharmaceutical formulation suitable for administration to a subject.

19. The method of any one of embodiments 1-18, wherein the polypeptide is a therapeutic polypeptide.

20. The method of embodiment 16, wherein the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragmentAntibody drug conjugates, thiomab ^TM Or Thiomab ^TM A drug conjugate.

21. The method of any of embodiments 1-20, wherein the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer.

22. The method of any of embodiments 1-21, wherein the mixed mode anion exchange chromatography material comprises a quaternary amine moiety.

23. The method of any of embodiments 1-22, wherein the mixed mode anion exchange chromatography material comprises a solid support.

24. The method of any of embodiments 1-23, wherein the mixed mode anion exchange chromatography material is contained in a column.

25. The method of any of embodiments 1-24, wherein the mixed mode anion exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material.

26. The method of any of embodiments 1-25, wherein the mixed mode anion exchange chromatography material is

MAX chromatography materials.

27. The method of any of embodiments 1-26 wherein the non-ionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD).

28. A method for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the steps of:

a) Applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising mobile phase a and mobile phase B, wherein mobile phase a comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an organic solvent solution of ammonium hydroxide;

b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step a);

c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step c);

d) Quantifying the nonionic surfactant.

29. The method of embodiment 28, wherein the organic solvent of mobile phase B is methanol.

30. The method of embodiment 28 or 29, wherein the ratio of mobile phase B to mobile phase a in step a) is about 10.

31. The method of any one of embodiments 28-30, wherein the ratio of mobile phase B to mobile phase a is increased to about 45.

32. The method of any one of embodiments 28-31, wherein the ratio of mobile phase B to mobile phase a is increased to about 100 in step c).

33. The method of any of embodiments 28-32, wherein mobile phase a comprises about 2% aqueous ammonium hydroxide.

34. The method of any of embodiments 28-33, wherein mobile phase B comprises about 2% ammonium hydroxide in methanol.

35. The method of any one of embodiments 28-34, wherein the flow rate for chromatography is about 1.4 mL/min.

36. The method of embodiment 35, wherein step b) begins about 1 minute after chromatography begins and ends about 4.4 minutes after chromatography begins.

37. The method of embodiment 35 or 36, wherein step c) begins about 4.5 minutes after the start of chromatography and ends about 7.6 minutes after the start of chromatography.

38. The method of any one of embodiments 28-37, wherein the non-ionic surfactant is a polysorbate.

39. The method of embodiment 38, wherein said polysorbate is polysorbate 20 or polysorbate 80.

40. The method of embodiment 38 or 39, wherein the concentration of polysorbate in said composition is in the range of about 0.001% to 1.0% (w/v).

41. The process of embodiment 28 wherein the organic solvent of mobile phase B is acetonitrile.

42. The method of embodiment 41, wherein the ratio of mobile phase B to mobile phase a in step a) is about 10.

43. The method of embodiment 41 or 42, wherein the ratio of mobile phase B to mobile phase a in step B) is increased to about 40.

44. The method according to any of embodiments 41 to 43, wherein the ratio of mobile phase B to mobile phase a is increased to 100 in step c).

45. The method of any of embodiments 41-44, wherein mobile phase A comprises an about 2% solution of ammonium hydroxide in water or in 43% methanol.

46. The method of any of embodiments 41-45, wherein mobile phase B comprises about 2% ammonium hydroxide in acetonitrile.

47. The method of any one of embodiments 41-46, wherein the non-ionic surfactant is a poloxamer.

48. The method of embodiment 48, wherein said poloxamer is poloxamer P188.

49. The method of embodiment 48 or 49, wherein the concentration of poloxamer in the composition is in the range of about 0.001% to 1.0% (w/v).

50. The method of any one of embodiments 28-49, wherein the composition further comprises N-acetyltryptophan and/or methionine.

51. The method of embodiment 50, wherein the concentration of N-acetyltryptophan in the composition ranges from about 0.1mM to about 10mM.

52. The method of embodiment 50, wherein the concentration of methionine in the composition ranges from about 0.1mM to about 100mM.

53. The method of any one of embodiments 28-52, wherein the concentration of the polypeptide in the composition is about 1mg/mL to about 250mg/mL.

54. The method of any one of embodiments 28-53, wherein the formulation has a pH of about 4.5 to about 7.5.

55. The method of any of embodiments 28-54, wherein the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents.

56. The method of any of embodiments 28-55, wherein said composition is a pharmaceutical formulation suitable for administration to a subject.

57. The method of any one of embodiments 28-56, wherein said polypeptide is a therapeutic polypeptide.

58. The method of embodiment 57, wherein the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, THIOMAB ^TM ，THIOMAB ^TM A drug conjugate.

59. The method of any of embodiments 28-58, wherein the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer.

60. The method of any of embodiments 28-59, wherein the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety.

61. The method of any of embodiments 28-60, wherein the mixed mode cation exchange chromatography material comprises a solid support.

62. The method of any of embodiments 28-61, wherein the mixed mode cation exchange chromatography material is contained in a column.

63. The method of any of embodiments 28-62, wherein the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material.

64. The method of any of embodiments 28-63, wherein the mixed mode cation exchange chromatography material is

MCX chromatography material.

65. The method of any of embodiments 28-64 wherein the non-ionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD).

Claims (38)

1. A method for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the steps of:

a) Applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is applied to the chromatography material in a solution comprising mobile phase a and mobile phase B, wherein mobile phase a comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an organic solvent solution of ammonium hydroxide;

b) Eluting the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step a);

c) Eluting the non-ionic surfactant from the chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step c);

d) Quantifying the nonionic surfactant.

2. The process of claim 1, wherein the organic solvent of mobile phase B is methanol.

3. The process according to claim 1 or 2, wherein the ratio of mobile phase B to mobile phase a in step a) is 10.

4. The method according to any one of claims 1 to 3, wherein the ratio of mobile phase B to mobile phase A is increased to 45.

5. The process according to any one of claims 1 to 4, wherein in step c) the ratio of mobile phase B to mobile phase A is increased to 100.

6. The method of any of claims 1-5, wherein mobile phase A comprises a 2% aqueous ammonium hydroxide solution.

7. The process of any of claims 1-6, wherein mobile phase B comprises a 2% solution of ammonium hydroxide in methanol.

8. The method of any one of claims 1-7, wherein the flow rate for chromatography is 1.4 mL/min.

9. The method of claim 8, wherein step b) begins 1 minute after chromatography begins and ends 4.4 minutes after chromatography begins.

10. The method of claim 8 or 9, wherein step c) begins 4.5 minutes after the start of chromatography and ends 7.6 minutes after the start of chromatography.

11. The method of any one of claims 1-10, wherein the non-ionic surfactant is a polysorbate.

12. The method of claim 11, wherein the polysorbate is polysorbate 20 or polysorbate 80.

13. The method of claim 11 or 12, wherein the concentration of polysorbate in the composition is in the range of 0.001% to 1.0% (w/v).

14. The process of claim 1, wherein the organic solvent of mobile phase B is acetonitrile.

15. The process of claim 14, wherein the ratio of mobile phase B to mobile phase a in step a) is 10.

16. The method of claim 14 or 15, wherein the ratio of mobile phase B to mobile phase a in step B) is increased to 40.

17. The method according to any one of claims 14 to 16, wherein the ratio of mobile phase B to mobile phase a in step c) is increased to 100.

18. The process of any of claims 14-17, wherein mobile phase a comprises a 2% solution of ammonium hydroxide in water or in 43% methanol.

19. The process of any of claims 14-18, wherein mobile phase B comprises a 2% ammonium hydroxide solution in acetonitrile.

20. The method of any one of claims 14-19, wherein the non-ionic surfactant is a poloxamer.

21. The method of claim 20, wherein the poloxamer is poloxamer P188.

22. The method of claim 20 or 21, wherein the concentration of poloxamer in the composition is in the range of 0.001% to 1.0% (w/v).

23. The method of any one of claims 1-22, wherein the composition further comprises N-acetyltryptophan and/or methionine.

24. The method of claim 23, wherein the concentration of N-acetyltryptophan in the composition ranges from 0.1mM to 10mM.

25. The method of claim 23, wherein the concentration of methionine in the composition ranges from 0.1mM to 100mM.

26. The method of any one of claims 1-25, wherein the concentration of the polypeptide in the composition is from 1mg/mL to 250mg/mL.

27. The method of any one of claims 1-26, wherein the formulation has a pH of 4.5 to 7.5.

28. The method of any one of claims 1-27, wherein the composition further comprises one or more excipients selected from the group consisting of stabilizers, buffers, and tonicity agents.

29. The method of any one of claims 1-28, wherein the composition is a pharmaceutical formulation suitable for administration to a subject.

30. The method of any one of claims 1-29, wherein the polypeptide is a therapeutic polypeptide.

31. The method of claim 30, wherein the therapeutic polypeptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody drug conjugate, THIOMAB ^TM ，THIOMAB ^TM A drug conjugate.

32. The method of any one of claims 1-31, wherein the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer.

33. The method of any of claims 1-32, wherein the mixed mode cation exchange chromatography material comprises sulfonic acid moieties.

34. The method of any one of claims 1-33, wherein the mixed mode cation exchange chromatography material comprises a solid support.

35. The process of any one of claims 1-34, wherein the mixed mode cation exchange chromatography material is contained in a column.

36. The method of any of claims 1-35, wherein the mixed mode cation exchange chromatography material is a High Performance Liquid Chromatography (HPLC) material.

37. The process of any one of claims 1-36, wherein the mixed mode cation exchange chromatography material is

MCX chromatography material.

38. The method of any one of claims 1-37, wherein the non-ionic detergent is quantified by Evaporative Light Scattering (ELSD) or by using a Charged Aerosol Detector (CAD).

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